WO2017004435A1

WO2017004435A1 - Methods of treating immune disorders

Info

Publication number: WO2017004435A1
Application number: PCT/US2016/040517
Authority: WO
Inventors: Lilian NOHARA; Wilfred Jefferies; Frank FENNINGER
Original assignee: Biommune Technologies Inc.
Priority date: 2015-06-30
Filing date: 2016-06-30
Publication date: 2017-01-05

Abstract

Provided are CaV1.4 agonists, compositions, and methods for increasing receptor editing, ameliorating cell death, and/or increasing immunocompetency in an immune cell or subject. Also included are methods for treating an immunocompromised subject or a subject with an autoimmune disease by administering a CaV1.4 agonist or composition to the subject.

Description

Methods of Treating Immune Disorders

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Application No. 62/186,850, filed June 30, 2015, which is incorporated by reference in its entirety.

STATEMENT REGARDING THE SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is BIMN_006_01WO_ST25.txt. The text file is about 50 KB, was created on June 30, 2016, and is being submitted electronically via EFS-Web.

BACKGROUND

Technical Field

The present disclosure relates generally to CaVl.4 agonists, compositions, and methods for increasing receptor editing, ameliorating cell death, and/or increasing immunocompetency in an immune cell or subject. Also included are methods for treating an immunocompromised subject or a subject with an autoimmune disease by administering a CaV1.4 agonist or composition to the subject. Description of the Related Art

B-lymphocytes, also known as B-cells, are lymphocytes involved with humoral immunity of the adaptive immune system. B-lymphocytes are distinguished from other lymphocytes by the presence of a B-cell receptor (BCR). This receptor allows a B-lymphocyte to bind to and initiate a response to a specific antigen. The principal functions of B-lymphocytes are to produce antibodies, to perform the role of antigen-presenting cells (APCs), and to develop into memory B-lymphocytes after activation by antigen interaction. B-lymphocytes also release cytokines to signal and regulate immune regulatory functions.

B-lymphocyte development is a strictly regulated process that occurs through several stages, each stage representing a change in the genome content at the antibody loci. Pre-pro B-lymphocytes develop into immature B-lymphocytes during which the recombination activating genes (RAG) proteins rearrange their immunoglobulin (Ig) genes to eventually express a B-Lymphocyte receptor (BCR).

Central tolerance mechanisms occur during the immature stage whereby the BCR is tested for reactivity against self- or auto-antigens. At this checkpoint, only B lymphocytes with a BCR that does not bind self- antigens will be selected and allowed to proceed into the periphery. B lymphocytes with self-reacting BCRs will either undergo apoptosis (clonal deletion), functional inactivation (anergy), or will attempt to rescue themselves by modifying their BCR. The latter process, termed receptor editing, gives a B- lymphocyte a second opportunity to rearrange its Ig genes to express a nonautoreactive BCR. Up to 50% of the B-lymphocytes leaving the bone marrow have undergone receptor editing, making this a crucial process of B-Lymphocyte development.

Receptor editing is considered to be a critical event during B-lymphocyte development and a major mechanism for preventing the emergence of self-reactive specificities, maintaining normal tolerance, and establishing BCR diversity. Increasing receptor editing may therefore be useful for treating immune-related disorders. For these reasons, a need in the art exists for agents that increase receptor editing.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present disclosure relate generally to methods for inducing receptor editing in a B-lymphocyte, comprising contacting the B-lymphocyte with a Cavl.4 agonist, thereby inducing receptor editing in the B-lymphocyte.

In some embodiments, the B-lymphocyte expresses Cavl.4. In certain embodiments, the B- lymphocyte is an immature B-lymphocyte or a transitional lymphocyte. In some embodiments, the B- lymphocyte expresses an autoreactive B cell receptor (BCR).

In certain embodiments, the receptor editing modifies sequences of light chain V and/or J genes. In certain embodiments, the receptor editing replaces Ig kappa light chain with Ig lambda light chain in the BCR.

In some embodiments, the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified mononucleotide, a lipopeptide, an antimicrobial, a small molecule, or a pharmaceutical molecule.

In some embodiments, the Cavl.4 agonist increases calcium conductance of Cavl.4. In some embodiments, the Cavl.4 agonist increases Cavl.4 expression in the cell. In some embodiments, the Cavl.4 agonist increases membrane localization of Cavl.4 in the cell. Also included are methods for ameliorating death in a B-lymphocyte, comprising contacting the cell with a Cavl.4 agonist, thereby ameliorating death in the B-lymphocyte, wherein the B-lymphocyte expresses an autoreactive B cell receptor (BC ).

In some embodiments, the immune cell expresses Cavl.4. In particular embodiments, the B- lymphocyte is an immature B-lymphocyte or a transitional lymphocyte.

In some embodiments, the Cavl.4 agonist stimulates receptor editing in the cell. In some embodiments, the Cavl.4 agonist increases calcium conductance of Cavl.4. In some embodiments, the Cavl.4 agonist increases Cavl.4 expression in the cell. In particular embodiments, the Cavl.4 agonist increases membrane localization of Cavl.4 in the cell. In specific embodiments, the cell death is apoptosis.

Also included are methods for increasing an amount of immunocompetent B-lymphocytes, comprising contacting bone marrow with a Cavl.4 agonist, wherein the bone marrow comprises B- lymphocytes, thereby increasing the amount of the immunocompetent B-lymphocytes. In some embodiments, the B-lymphocytes are immature B-lymphocytes and/or transitional B-lymphocytes. In some embodiments, a subset of the B-lymphocytes expresses an auto-reactive BCR. In certain embodiments, the subset of the B-lymphocytes express Cavl.4.

In particular embodiments, contacting the bone marrow with the Cavl.4 agonist stimulates receptor editing in the subset of the B-lymphocytes. In some embodiments, contacting the bone marrow with the Cavl.4 agonist ameliorates cell death in the subset of the B-lymphocytes. In some

embodiments, the cell death is apoptosis. In some embodiments, contacting the bone marrow with the Cavl.4 agonist reduces an amount of transitional B-lymphocytes that leave the bone marrow and express an autoreactive BCR.

In some embodiments, the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified mononucleotide, a lipopeptide, an antimicrobial, a small molecule, or a pharmaceutical molecule. Certain embodiments relate to methods for increasing an amount of immunocompetent B- lymphocytes in a subject, comprising administering to the subject a Cavl.4 agonist, thereby increasing the amount of the immunocompetent B-lymphocytes in the subject. In some embodiments, the subject is a mammal. In specific embodiments, the subject is a human.

In some embodiments, administering to the subject the CaV1.4 agonist reduces an amount of chronically activated B-lymphocytes in the subject. In some embodiments, administering to the subject the CaV1.4 agonist reduces an amount of anergic B-lymphocytes in the subject. In some embodiments, administering to the subject the CaV1.4 agonist reduces an amount of B-lymphocytes that express an autoreactive BC . In some embodiments, administering to the subject the CaV1.4 agonist increases an amount of cells that undergo receptor editing, wherein the cells that undergo receptor editing are B- lymphocytes that express an autoreactive BCR.

Also included are methods for treating an autoimmune disease in a subject, comprising administering to the subject a Cavl.4 agonist. In some embodiments, the subject is a mammal. In particular embodiments, the subject is a human. In particular embodiments, the subject produces an autoantibody. In some embodiments, administering to the subject the Cavl.4 agonist reduces autoantibody in the subject.

In certain embodiments, the autoimmune disease is Addison's disease, Celiac Disease, Dermatomyositis, Grave's Disease, Hashimoto's Thyroiditis, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia, Reactive Arthritis, Rheumatoid Arthritis, Sjogren Syndrome, Systemic Lupus Erythematosus, Type I Diabetes.

Also included are methods for treating immunodeficiency in a subject, comprising administering to the subject an agonist of Cavl.4. In some embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In some embodiments, the immunodeficiency is a primary immunodeficiency. In some embodiments, the immunodeficiency is an acquired immunodeficiency. In particular embodiments, the subject has undergone cancer chemotherapy, organ transplantation, marrow transplantation, tissue transplantation, or glucocorticoid therapy. In some embodiments, the immunodeficiency is a humoral immune deficiency. In some embodiments, the subject has Multiple Myeloma, Chronic Lymphoid Leukemia, AIDS, Lymphoma, or Chronic Granulomatous Disease.

In some embodiments, the subject has or is at risk of having at least one opportunistic infection. In particular embodiments, the at least one opportunistic infection is selected from the list consisting of Acinetobacter baumanni, Aspergillus sp., Candida albicans, Clostridium difficile, Cryptococcus neoformans, Cryptosporidium sp., Cytomegalovirus, Geomyces destructans, Giardia intestinalis,

Histoplasma capsulatum, Hemophilus influenza, Isospora belli, Polyomavirus JC polyomavirus, Kaposi's Sarcoma caused by Human herpesvirus 8 (HHV8), Kaposi's sarcoma-associated herpesvirus (KSHV), Legionnaires' Disease (Legionella pneumophila), Microsporidium, Mycobacterium avium complex (MAC) (Nontuberculosis Mycobacterium), Pneumocystis jirovecii, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, Streptococcus pyogenes, or Toxoplasma gondii.

In certain embodiments, the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, NAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified mononucleotide, a lipopeptide, an antimicrobial, a small molecule, or a pharmaceutical molecule.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows that CaV1.4-deficent mice have altered peripheral B-lymphocyte development, (a) Frequencies (percentage of lymphocytes) and total numbers of B-lymphocyte in the bone marrow were determined by flow cytometry analysis, (b) Total numbers are shown for each B-lymphocyte subset present in the bone marrow, (c) Frequencies and total numbers of B-lymphocytes in the spleen are shown, (d) Frequencies and total numbers are shown for each B-lymphocyte subset present in the spleen, (e) Frequency of B-lymphocytes in the peritoneal cavity are shown, (f) Frequency is shown for each B-lymphocyte subset present in the peritoneal cavity. Error bars represent the standard deviation (SD); * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 2 shows results of flow cytometry quantifying B-lymphocytes at different stages of B- lymphocyte development in lethally irradiated congenic CD45.1+ wild-type recipient mice intravenously injected with a 1:1 mixture of wild-type CD45.1+CD45.2+ (competitor) plus wild-type or Cacnalf CD45.2+ (donor) bone marrow 8 weeks after reconstitution. Results are presented as the ratio of donor to competitor lymphocytes (+/+squares, wild-type; -/- triangles, Cacnalf^7") in (a) bone marrow, (b) spleen, and (c) peritoneal cavity. Error bars represent SD.

Figure 3 shows results of flow cytometry of wild-type (+/+, top line) and Cacnalf^' (-/-, bottom line) splenocytes loaded with the intracellular Ca²⁺ dyes Fluo-4 and FuraRed. The intracellular Ca²⁺ levels in splenic B-lymphocytes are plotted as the ratio of Fluo-4/FuraRed over time, (a) Splenic B-lymphocytes were stimulated with anti-lgM (BCR) and then with ionomycin (Ion) at the indicated times (arrows), (b) Splenic B-lymphocytes were stimulated with anti-lgM (BCR) in the absence of free extracellular Ca²⁺ and then free extracellular Ca²⁺was added back at the indicated time points, (c) Splenic B-lymphocytes were stimulated with thapsigargin (Tg) and then extracellular Ca²⁺ was chelated by EGTA addition, (d) The basal intracellular Ca²⁺ levels of follicular B lymphocytes are shown as fold difference of Fluo-4 staining. Significance was assessed by unpaired t-test; ** p<0.005.

Figure 4 shows that CaVl.4 mediates Ca²⁺ entry across the plasma membrane of splenic B- lymphocytes, (a) Sample traces of inward barium currents recorded on wild-type (+/+, n = 7) and Cacnalf^' (-/-, n = 6) splenic B-lymphocytes following BCR activation are shown, (b) Current density comparison at +10 mV between wild-type (n=7) and Cacnalf^' (n=6) splenic B-lymphocytes is shown, (c) Comparison of inward barium currents from wild-type splenic B-lymphocytes (n = 4) before and after treatment with 10 μΜ of nifedipine (-Nif and +Nif, respectively) is shown, (d-e) Sample l-V relationships are shown of wild-type B-lymphocyte inward current recorded with (d) normal or (e) modified (+ Ba²⁺) TEA internal solutions in the presence of 4 μΜ of Bay K8644 in the external solution with a ramp pulse protocol following BCR activation. The black lines indicate modified Boltzmann equation fits to the l-V relationships, (f) A comparison is shown of reversal potential between cells recorded with normal internal solution (n = 6) and cells recorded with modified internal solution (n = 5). Error bars represent the standard error of the mean (SEM); *p < 0.05.

Figure 5 shows surface expression of (a) CD69 and (b) CD86 of wild-type (+/+, solid line; top panels) and Cacnalf ^' {-I- , solid line; bottom panels) splenocytes that are unstimulated (grey) or stimulated with anti-lgM or LPS at the indicated concentrations for 24h, as analyzed by flow cytometry. Numbers above bracketed lines represent the percentage of splenic B-lymphocytes (B220+) that have upregulated the surface marker, (c) Flow cytometry analysis is shown of CFSE-labeled wild-type (+/+, solid line; top panels) and Cacnal ^' (-/- , solid line; bottom panels) splenocytes left unstimulated (grey) or stimulated with anti-lgM or LPS at the indicated concentrations for 72h. Numbers above bracketed lines represent the percentage of dividing cells, (d) Freshly isolated splenocytes were stained to identify follicular B lymphocytes (B220+ CD21+ CD23+). The histograms show the expression of the activation markers CD86, MHCII as well as the side scatter (SSC) of the follicular B lymphocytes.

Figure 6 shows CaV1.4-deficient splenic B-lymphocytes show reduced expression of B cell activating factor (BAFF) receptor and lower survival rates in response to BAFF. (a) Flow cytometry analysis of surface expression of BAFF receptors in total splenic B-lymphocytes and in splenic B cell subsets from wild-type {+/+, black) and Cacnalf^1' (-/-, grey) mice, (b) Percentage of live purified splenic B-lymphocytes from wild-type {+/+, squares) and Cacnalf^1' (-/-, triangles) mice cultured in the presence of the indicated concentrations of recombinant mouse BAFF for 72 h, assessed by flow cytometry. Error bars represent the SD; * p < 0.05 and ** p < 0.01.

Figure 7 shows antibody responses after immunization with TNP-Ficoll. Levels of specific antibodies elicited after the immunization of wild-type (n = 5) and Cacnalf^' (n = 5) were determined by ELISA. (a) TNP-specific anti-lgM and (b) anti-lgG3 antibody responses on day 0 (wild-type, +/+ black line; Cacnalf -/- grey line) and on day 7 after immunization (wild-type, +/+ squares; Cacnalf -/- circles) are shown. Error bars represent the SD.

Figure 8 shows results of (a) anti-DNA and (b) anti-nuclear ELISA analysis of the sera of young (8 weeks old) and old (40 weeks old) mice. The graph shows the OD450s measured for each mouse (n>=6 / group), (c) Follicular B-lymphocytes were stimulated with LPS and cultured for 24 hours. The concentrations of IFN-g, TNF and IL-6 where then determined using cytometric beads, (d) Albumin was quantified in the urine of 70 weeks old mice using Albustix Strips (n>=3 / group). Significance was assessed by unpaired t-test; **P<0.005; *P<0.05; ns P>=0.05.

Figure 9 shows results flow cytometry analysis of B220+ cells from the bone marrow of wild- type and Cacnalf^ mice that were stimulated with different concentrations of ct-lgM for 20 hours. The graph shows the expression of the IgK and IgA light chain of immature/transitional B lymphocyte s (B220+ CD93- IgDint). All data are representative of three independent experiments.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. All of the patent and non-patent literature references listed herein are incorporated by reference in their entireties.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

As used herein, the term "amino acid" is intended to mean both naturally occurring and non- naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4- hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or ) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the ε-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

The term "biological sample" includes a biological material that can be collected from a subject and used in connection with diagnosis or monitoring of biological states. Biological samples can include clinical samples, including body fluid samples, such as body cavity fluids, urinary fluids, cerebrospinal fluids, blood, and other liquid samples of biological origin; and tissue samples, such as biopsy samples, tumor or suspected tumor samples, and other solid samples of biological origin. Biological samples can also include those that are manipulated in some way after their collection, such as by treatment with reagents, culturing, solubilization, enrichment for certain biological constituents, cultures or cells derived therefrom, and the progeny thereof.

The term "conjugate" includes an entity formed as a result of covalent or non-covalent attachment or linkage of an agent or other molecule, e.g., a detectable entity, a biologically active molecule, PEG or other polymer, to an antibody described herein.

A "control" such as a "control subject" or "control tissue" includes a healthy subject or a healthy tissue sample, for example, which is not pathological or diseased. In certain embodiments, a control includes a non-diseased tissue from a different, healthy subject or the same subject being tested or diagnosed. A control can also include a reference standard, for example, a standard value generated from one or more healthy subjects or tissues.

As used herein, the terms "function" and "functional" and the like refer to a biological, enzymatic, or therapeutic function.

An "effective amount" is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition depends on the composition selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions of the invention can include a single treatment or a series of treatments.

"Homology" refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research. 12, 387-395, 1984), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated peptide" or an "isolated

polypeptide" and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances. In particular embodiments, the isolated polypeptide is an antibody.

A "increased" or "enhanced" amount is typically a "statistically significant" amount, and may include, for example, a 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increase. An increased or enhanced amount may also include a 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20-fold, 30 fold, 40 fold, 50 fold, 60 fold 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 1,000 fold, 10,000 fold, or greater than 10,000 fold increase (including all integers and ranges in between) relative to a control. Other examples of comparisons and "statistically significant" amounts are described herein. "Increase," as used herein, can refer to "agonize,"

"enhance," "inflate," "escalate," expand," "augment," "enlarge," or "raise."

A "decreased" or "reduced" amount is typically a "statistically significant" amount, and may include, for example, a 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease (including all integers and ranges in between) relative to a control. Other examples of comparisons and "statistically significant" amounts are described herein. "Decrease," as used herein, can refer to "inhibit," "reduce," "curb," "abate," "diminish," "lessen," "lower," or "weaken."

"Stabilization" as used herein, refers to increasing the half life of the functional form of a biological or pharmaceutical entity. Stabilization may be achieved, for example, by reducing the probability the entity will be degraded, or by increasing the probability that the entity will be in a functional or an active confirmation or state.

In certain embodiments, the "purity" of any given agent (e.g., a pharmaceutical compound) in a composition may be specifically defined. For instance, certain compositions may comprise an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, including all decimals in between, as measured, for example, and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. The polypeptides described herein are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. The polypeptides described herein may also comprise post-expression

modifications, such as glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence, fragment, variant, or derivative thereof.

The term "reference sequence" refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.

The terms "sequence identity" or, for example, comprising a "sequence 50% identical to," as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more)

polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin

Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wl, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example, disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15.

By "statistically significant," it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less. A "subject," as used herein, includes any animal that exhibits a symptom or condition, or is at risk for or suspected of exhibiting a symptom or condition, which can be diagnosed with an antibody described herein. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

A "subject subpopulation" or "patient subpopulation," as used herein, includes a subject or patient subset characterized as having one or more distinctive measurable and/or identifiable characteristics that distinguishes the subject or patient subset from others in the broader disease category (e.g., cancer) to which it belongs. Such characteristics include disease subcategories, gender, lifestyle, health history, organs/tissues involved, treatment history, etc. In some embodiments, a patient or subject subpopulation is characterized by the (e.g., reduced) amount or levels of CaV1.4 polypeptide in a biological sample, for example, a tumor sample.

"Substantially" or "essentially" means nearly totally or completely, for instance, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

"Substantially free" refers to the nearly complete or complete absence of a given quantity for instance, less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of some given quantity. For example, certain compositions may be "substantially free" of cell proteins, membranes, nucleic acids, endotoxins, or other contaminants.

"Treatment" or "treating," as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.

"Anergy" as used herein, is meant to mean "clonal anergy" or "lymphocyte anergy," and refers to the failure of clones of T or B-lymphocytes to react to antigen. Anergy can serve as a mechanism of maintaining immunologic tolerance to self.

"Antigen" as used herein, refers to a molecule that binds to an antibody or BC . "Self-antigen" or "autoantigen" as used herein, refers to any molecule or chemical group of an organism which is recognized as an antigen by an antibody, BCR, and/or a T-cell receptor.

"B-lymphocyte" as used herein, can be used interchangeably with "B-cell" and refers to a type of lymphocyte of the adaptive immune system. B-lymphocytes are the only cell type that can produce antibody and are therefore the central cellular component of humoral immune responses. B- lymphocytes can be distinguished from other lymphocytes, such as T cells and natural killer cells (NK cells), by the presence of a protein on the B-lymphocyte's outer surface known as a B-cell receptor (BCR). This specialized receptor protein allows a B cell to bind to a specific antigen. As used herein, "B- lymphocyte" or B-cell" can refer to a B-lymphocyte at any developmental stage as well as a mature B- lymphocyte.

"B-cell receptor," and "BCR" is used interchangeably with "B-lymphocyte antigen receptor complex" and as used herein refers to a multiprotein complex expressed on the surface of B- lymphocytes that recognizes antigen and transduces activating signals into the cell. The BCR includes membrane Ig, which is responsible for binging antigens, and Ig alpha and Ig beta, which initiate signaling events.

As used herein, the term "self-reactive" refers to the quality of specifically binding a self- antigen, and may describe a BCR that specifically binds a self-antigen or a B-lymphocyte that expresses a BCR that specifically binds a self-antigen.

"Immunocompetent" as used herein refers to the capability of having a normal immune response. As used herein, an immunocompetent B-lymphocyte is a mature B-lymphocyte that is non- self-reactive and is capable of producing an immune response to an antigen.

B-lymphocytes and receptor editing

B-lymphocytes proceed through a series of developmental stages to acquire a functional, non- self reactive antigen receptor (the B cell receptor, or BCR). Each stage represents a change in the genome content at the antibody loci of the BCR. The antigen-binding portion of the BCR includes immunoglobulin (Ig) heavy and light chain polypeptides. Both types of polypeptides contain an amino- terminal domain which directly contacts antigen and exhibits great sequence variability, and one or more constant domains.

The principal events during the maturation of B-lymphocytes are rearrangement and expression of Ig genes in a precise order, proliferation of immature cells, and selection of the mature B- lymphocytes. The maturation of B-lymphocytes from committed precursors occurs in the fetal liver and, after birth, in the bone marrow. During this process, B-lymphocyte progenitors develop into cells that express membrane bound IgM and IgD molecules and then leave the bone marrow to populate peripheral lymphoid organs, where they use their membrane Ig to recognize and respond to foreign antigens. During their maturation, cells of the B-lymphocyte lineage go through sequential stages, each of which is characterized by a specific pattern of Ig gene expression and the expression of other cell surface proteins that serve as phenotypic markers of these stages.

The exon encoding the variable domain of the heavy chain, but not the constant domain, is assembled from arrays of component variable (V), diversity (D), and joining (J) gene segments by site- specific DNA rearrangement. This rearrangement process, called VDJ recombination, underlies the diversity of BCRs and is evolutionarily conserved. The three segments recombine randomly to produce a unique variable domain in the BCR of each individual B-lymphocyte. Similar rearrangements occur for light-chain 'V region, except there are only two segments involved: V and J. VDJ and VJ recombination proceed through two distinct phases in which DNA double strand breaks (DSBs) are first introduced by the RAG proteins (the cleavage phase), and then subsequently repaired via the non-homologous end joining (NHEJ) pathway (the joining phase). In the cleavage phase, the RAG proteins catalyze DSBs at the ends of antigen receptor gene segments, directed by flanking recombination signal sequences (RSSs) containing a conserved heptamer and nonamer sequence element separated by spacer DNA either 12 or 23 bp in length. RAG-mediated cleavage generates two distinct DNA ends: blunt, 5'phosphorylated signal ends terminating at the heptamer and coding ends covalently sealed in DNA hairpin structure. In the joining phase, the hairpinned coding ends are first resolved and rendered accessible to enzymes that remove nucleotides or add them. Finally, ubiquitous factors involved in the NHEJ pathway are recruited to reorganize and seal the DNA ends. As a result, pairs of coding segments and their associated RSSs become transposed to form coding joints and signal joints, respectively. Signal joints typically contain two RSSs fused heptamer-to-heptamer, but coding joints are often imprecise, containing sequence deletions or insertions bearing palindromic repeats and/or nontemplated nucleotides.

The developmental stages through which B-lymphocytes mature are characterized by an ordered series of VDJ and VJ recombination events and selection processes that, if successfully completed, give rise to mature, non-self-reactive B-lymphocytes. Early in B-lymphocyte development, the immunoglobulin heavy chain locus undergoes VDJ rearrangement. The rearranged gene is functionally tested through the attempted pairing of the expressed μ heavy chain with the invariant polypeptides λ5 and Vpre-B (including the surrogate light chain, ψί). Successful pairing leads to the surface expression of the complex (termed the pre-B cell receptor), heavy chain allelic exclusion, down- regulation of the VDJ recombinase, closure of the heavy chain locus, and cell proliferation. The cells then exit cell cycle and enter a developmental stage during which the V(D)J recombinase is upregulated and light chain gene rearrangement ensues. Like the heavy chain gene, the rearranged light chain gene is functionally tested by pairing the expressed light chain with the μ heavy chain. Successful pairing leads to the expression of IgM on the cell surface, the phenotypic hallmark of an immature B cell. At this time, the cell begins to migrate out of the bone marrow, into the blood stream, and then to the spleen. As it does so, surface IgD begin to appear through alternative splicing of the heavy chain RNA transcript. During this transitional period, the BCR is tested for self-reactivity.

There are different outcomes for immature B-lymphocytes whose BCRs recognize self-antigen. These include clonal deletion, where the self-reactive B lymphocyte undergoes apoptosis, anergy, where the self reactive B-lymphocyte no longer responds to antigens, and receptor editing, where the B- lymphocyte alters the specificity of its BCR. Receptor editing involves reactiviation of the RAG genes, additional light chain recombination, and new Ig light chain production, which allows the B-lymphocyte to express a new BCR that is not self-reactive. B-lymphocytes reinitiate V(D)J recombination in order to edit receptor specificity away from auto-reactivity. Most often, the receptor editing process involves either the replacement of the offending light chain variable ex on or kappa deletion to promote λ light chain rearrangement. Rarely, heavy chain gene replacement may occur which involves recombination at a cryptic heptamer embedded in the 3' end of the rearranged VH segment. Receptor editing may therefore be considered a salvage pathway for potentially auto-reactive B-lymphocytes.

CaVl channels

CaVl.4 is a voltage-gated calcium channel. Voltage-gated calcium channels mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression in many different cell types. Voltage-gated calcium channels couple electrical events that alter membrane potential at the cell surface to physiological processes in the cells. Voltage-gated calcium channels are members of a gene superfamily of transmembrane ion channel proteins that includes voltage-gated potassium and sodium channels.

Calcium currents recorded in different cell types have diverse physiological and pharmacological properties, and so voltage-gated calcium channels are grouped based on the properties of their respective currents. In general, the CaVl family of voltage-gated calcium channels (also referred to as L- type voltage-gated calcium channels) open in response to a strong depolarization and produce long- lasting calcium current. CaVl channels are blocked by organic CaVl channel antagonists, including dihydropyridines, phenylalkylamines, and benzothiazepines. CaVl channels generate the main calcium currents recorded in muscle and endocrine cells, where they initiate contraction and secretion. Currents from CaVl channels activating at lower voltages also exist predominantly in neurons and cardiac pacemaker cells. CaVl channels are also expressed in cells that are not considered excitable, including in hematopoietic cells such as T-lymphocytes and B-lymphocytes.

The CaVl channels are complex proteins composed of four or five distinct subunits that are encoded by multiple genes. The ctl subunit of 190 to 250 kDa is the largest subunit, and it incorporates the conduction pore, the voltage sensor and gating apparatus, and most of the known sites of channel regulation by secondary messengers, drugs, and toxins. A particular CaVl channel will take its name (e.g. Cavl.4) from the alpha 1 subunit it contains. Like the alpha subunits of sodium channels, the alpha 1 subunit of a voltage gated calcium channel is organized in four homologous domains (l-IV), also called motifs, with six transmembrane segments (S1-S6) in each. The transmembrane segments of each domain are numbered in the order they are arranged from the N-terminal to the C-terminal.

The alpha 1 subunit forms the channel structure of the voltage-gated calcium channel protein. The pore of the calcium channel is formed at the center of a pseudo-symmetric arrangement of the four domains, and the pore loops between S5 and S6 of each domain form the narrow, extracellular end of the channel. These pore loops determine ion conductance and selectivity, and changes of only three specific amino acids in the pore loops in domains I, II I, and IV will convert a channel's selectivity from calcium ions to sodium ions. The S4 segments of each domain serve as the channel's voltage sensor. An intracellular beta subunit and a transmembrane, disulfide-linked alpha2 beta subunit complex are components of most types of calcium channels. A gamma subunit has also been found in skeletal muscle calcium channels, and related subunits are expressed in heart and brain. Although these auxiliary subunits modulate the properties of the channel complex, the pharmacological and electrophysiological diversity of calcium channels arises primarily from the ctl subunits.

The opening and closing of the voltage-gated calcium channels are primarily gated by changes in membrane potential, which cause movement of charges across the membrane and drive conformational changes that open and close the pore. The positively charged S4 segments are thought to undergo outward and rotational movement through the protein structure during the gating process, as proposed in the 'sliding helix' and 'helical screw' models of gating (Reviewed in Catterall et al, (2007) Toxicon 49(2), pp 124-141). This structure suggests that the pore is closed at its intracellular end and discriminates ions at the narrow ion selectivity filter at its extracellular end.

L-type voltage-gated calcium channels are found in populations of cells considered to be excitable. Excitable cells are cells where specific stimulations can trigger changes in the membrane potential globally throughout the cell or locally in a region of the cell. Calcium channels can function to regulate the changes in membrane potential. Additionally, calcium ions that enter the cytosol when calcium channels are open can act as a secondary messenger that can continue to regulate cellular processes after the depolarization event.

As used herein, the terms "L-type Voltage-Gated Calcium Channel" and "CaVl" channels are used interchangeably, and are meant to include Cavl.l, Cavl.2, Cavl.3, and Cavl.4 unless otherwise specified. "L-type Voltage-Gated Calcium Channel" and "CaVl" channels also comprise Cavl.l, Cavl.2, Cavl.3, and Cavl.4 channels that may undergo variations in post-expression modifications, such as glycosylations, acetylations, phosphorylations and the like, and include the entire Cavl.l, Cavl.2, Cavl.3, and Cavl.4 proteins, as well as subsequences, fragments, variants (including but not limited to variants resulting from alterative splicing), or derivatives thereof.

Cavl.4

At least four subtypes of CaVl channels have been described. These subtypes are categorized by the alpha 1 subunits they contain, which are each encoded by separate genes. The gene encoding Cavl.4 is CACNA1F. High levels of Cavl.4 are found in retina, spleen, thymus, and bone marrow. Data from mouse and human studies demonstrate that each CaVl channel subtype is found in immune cells (Reviewed in Omilusik et al (2013) Frontiers in Immunology, vol 4:164). Cavl.4 expression has been observed in the human Jurkat T cell line, as well as in human and rat spleen and thymus, and human and mouse T cells. CaVl.4 is also present in B-lymphocytes.

Certain embodiments contemplate a model whereby CaVl.4 signaling contributes to auto- antigen induced BCR-crosslinking to prompt receptor editing. Without being bound by theory, particular embodiments contemplate that the binding of self-antigen to the BCR of an immature or transitional B- lymphocyte triggers CaV1.4-dependent Ca²⁺ influx. This CaV1.4-dependent Ca²⁺ influx increases the probability that the self-reactive B-lymphocyte will undergo receptor editing and reduces the probability that the cell will become anergic or undergo apoptosis. Receptor editing alters the specificity of the BCR away from self-reactivity. Therefore, certain embodiments contemplate that CaV1.4 channel activity reduces the amount of self-reactive B-lymphocytes and reduces the amount of B-lymphocytes that undergo apoptosis or become anergic, thereby maintaining the pool of immature or transitional B- lymphocytes that develop into mature, immunocompetent B-lymphocytes.

Without being bound by theory, particular embodiments contemplate a model whereby reducing or ablating CaV1.4-dependent Ca²⁺ signaling will reduce the amount of self-reactive immature or transitional B-lymphocytes that undergo receptor editing. Particular embodiments contemplate that the deficiencies in receptor editing caused by impaired CaV1.4 signaling will lead to an inflammatory state in peripheral mature B-lymphocytes. Some embodiments contemplate that after failed receptor editing due to a reduction or ablation of CaV1.4 activity, most auto-reactive B-lymphocytes will undergo apoptosis before they can migrate to the periphery, resulting in lower numbers of mature,

immunocompetent B-lymphocytes. Further, when CaV1.4 activity is reduced or ablated, remaining mature B-lymphocytes will be chronically activated, will have elevated internal Ca²⁺ levels, and show decreased Ca²⁺ mobilization across the cell membrane. Particular embodiments contemplate that when CaV1.4 activity is reduced or ablated, a portion of the remaining mature B-lymphocytes will have an anergic phenotype, and another portion of the remaining mature B-lymphocytes will be auto-reactive cells left escaping immune tolerance. Particular embodiments contemplate that when CaV1.4 is chronically impaired or ablated in a subject, production of autoantibodies will increase as the subject ages.

Certain embodiments contemplate a model whereby increasing CaV1.4 activity increases the number of self-reactive immature or transitional B-lymphocytes that undergo receptor editing.

Particular embodiments contemplate that increasing the amount of self-reactive B-lymphocytes that undergo receptor editing will reduce the number of self-reactive B-lymphocytes that undergo apoptosis or become anergic. This model contemplates that a majority of self-reactive B-lymphocytes that undergo receptor editing will alter the specificity of their BCR and become non-self reactive, thus increasing the pool of immature B-lymphocytes that can mature into immunocompetent B-lymphocytes. Therefore, this model predicts that increasing receptor editing through CaV1.4 activation increases the amount of mature, circulating, immunocompetent B-lymphocytes. Certain embodiments contemplate that increasing CaV1.4 activity will reduce the amount of B-lymphocytes in an inflammatory state.

Particular embodiments contemplate that increasing CaV1.4 activity will reduce the amount of B- lymphocytes that are chronically activated. Some embodiments contemplate that increasing CaV1.4 activity will increase the amount of mature B-lymphocytes that are immunocompetent.

CaV1.4 has several splice variants. Alternative splicing is a regulated process during gene expression that results in a single gene coding for multiple isoforms of the protein. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. Consequently the proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence. Alternative splicing occurs as a normal phenomenon in eukaryotes, and has been described in the genes encoding L-type voltage-gated subunits. As used herein, the term CaVl channel," unless otherwise specified, includes Cavl.l, Cavl.2, Cavl.3, and Cavl.4, and all isoforms of the alpha 1 subunit that result from alternative splicing of mRNA encoding Cavl.l, Cavl.2, Cavl.3, and Cavl.4. Likewise, as used herein the term "CaV1.4" refers to a channel containing a CaV1.4 alpha subunit, and includes all isoforms of CaV1.4 that result from alternative mRNA splicing unless specified otherwise.

The primary amino acid sequence of human and mouse CaV1.4 channel alpha 1 subunits are shown in Table 1 below. Of note, mRNAs encoding alpha 1 subunits of L-type voltage-gated calcium channels have splice variants that can result in different isoforms of the polypeptide. Therefore, the amino acid sequences listed in table 1 are exemplary.

gylgrssgplrtftclhvpgthsdpshgkrgsadslveavliseglglf ardprfvalakqeiadacrltldemdnaasdllaqgtsslysdeesils rfdeedlgdemacvhal

Mouse CACNA1F msesevgkdttpepspangtgpgpewglcpgpptvgtdtsgasglgtpr 2 CaVl .4 rrtqhnkhktvavasaqrspralfcltltnpirrscisivewkpfdili

litifancvalgvyipfpeddsntanhnleqveyvflviftvetvlkiv ayglvlhpsayirngwnlldfiivvvglfsvlleqgpgrpgdaphtggk pggfdvkalrafrvlrplrlvsgvpslhivlnsimkalvpllhiallvl fviiiyaiiglelflgrmhktcyfigsdmeaeedpspcassgsgrset1 nhtecrgrwpgpnggitnfdnfffamltvfqcitmegwtdvlywmqdam gyelpwvyfvslvifgs ffvlnlvlgvlsgefskerekakargdfqklr ekqqmeedlrgyldwitqaeeldlhdpsvdgnlaslaeegraghrpqls eltnrrrgrlrwfshstrsthstsshaslpasdtgsmtdtpgdedeeeg tmasctrclnkimktricrhfrranrglrarerravksnacywavlllv flntltiasehhgqplwltqtqeyankvllclftvemllklyglgpsvy vas ffnrfdefvvcggilettlvevgamqplgisvlrcvrllrifkvtr hwaslsnlvasllnsmksias11111 fIfiiifsllgmqlfggkfnfdq thtkrstfdtf qalltvfqiltgedwnvvmydgimayggpffpgmlvc vyfiilficgnyillnvflaiavdnlasgdagtakdkgrekssegnppk enkvlvpggenedakgarsegaapgmeeeeeeeeeeeeeeeeengaghv ellqevvpkekvvpipegsaffclsqtnplrkachtlihhhiftslilv fiilssvslaaedpirahs frnhilgyfdyaftsiftveillkmtvfga flhrgs fcrswfnlldllvvsvslis fgihssaisvvkilrvlrvlrpl rainrakglkhvvqcvfvairtignimivttllqfmfacigvqlfkgkf ysctdeakhtlkeckgs fliypdgdvsrplvrerlwvnsdfnfdnvlsa mmalftvstfegwpallykaidanaedegpiynyhveisvffivyiiii affmmnifvgfviitfraqgeqeyqnceldknqrqcveyalkaqplrry ipknphqyrvwatvnsaafeylmfllillntvalamqhyeqtapfnyam dilnmvftglftiemvlkiiafkpkhyfadawntfdalivvgsvvdiav tevnngghlgessedssrisitffrlfrvmrlvkllskgegirtllwtf iks fqalpyvalliamiffiyavigmqmfgkvalqdgtqinrnnnfqtf pqavlllfrcatgeawqeimlaslpgnrcdpesdfgpgeeftegss fai vyfis ffmlcafliinlfvavimdnfdyltrdwsilgphhldefkriws eydpgakgrikhldvvallrriqpplgfgklcphrvackrlvamnvpln sdgtvtfnatlfalvrtslkiktegnldqanqelrmvikkiwkrikqkl ldevipppdeeevtvgkfyatfliqdyfrkfrrrkekgllgreaptsts salqaglrslqdlgpeirqaltydteeeeeeeeavgqeaeeeeaennpe pykdsidsqpqsrwnsrisvslpvkeklpdslstgpsdddglapnsrqp sviqagsqphrrssgvfmftipeegsiqlkgtqgqdnqneeqevpdwtp dldeqagtpsnpvllpphwsqqhvnghhvprrrllpptpagrkps ftiq clqrqgscedlpipgtyhrgrtsgpsraqgswaappqkgrllyaplllv eestvgegylgklggplrtftclqvpgahpnpshrkrgsadslveavli seglglfaqdprfvalakqeiadachltldemdsaasdllaqrttslys deesilsrfdeedlgdemacvhal

The exemplary mRNA sequences of human and mouse CaVl.4 channel alpha subunits are shown in Table 2 below. Of note, mRNAs encoding CaV1.4 channel alpha subunits have splice variants that can result in different isoforms of the polypeptide. Therefore, the amino acid sequences listed in table 2 are exemplary. In some embodiments, the invention contemplates the use of splice variants and isoforms.

Table 2 : CaVl 4 Nucleotide Sequences

Name Gene Nucleotide Sequence SEQ Symbol ID

NO:

Human CACNA1 F ctccaaagctgggggaagagaggggggttgtgtgcagatggcccttcaatctcgaaagaa 3 CaVl. agatgtcggaatctgaaggcgggaaagacaccaccccagagcccagtccagccaatgggg

4 caggccctggtcccgaatgggggctgtgccccgggcccccagctgtggaaggtgaaagca

gtggggcatcaggcctagggacccctaagcgaagaaaccagcacagcaagcacaagacag tggcagtggccagtgcccagcggtcacctcgggcactcttctgcctcaccctggccaatc ctctgcgacggtcctgcatcagcatcgtggagtggaagcccttcgacatcctcatcctgc tgaccatctttgccaactgcgtggccctgggagtttacatccccttccctgaggacgact ccaacactgccaaccacaacctggagcaggtggagtacgtattcctggtgattttcactg tggagacggtgctcaagatcgtggcctacgggctggtgctccaccccagcgcctacatcc gcaatggctggaacctactcgacttcatcatcgtcgtggtcgggctgttcagcgttctgc tggagcagggccccggacggccaggcgacgccccgcacaccgggggaaagccaggaggct tcgatgtgaaggcattgagggcgtttcgggtgctgcggccactgaggctggtgtctgggg tcccgagcctgcacatagtgctcaattccatcatgaaggctctggtgccgctgctgcaca ttgcactgctcgtgctcttcgtcatcatcatttatgccatcattgggctcgagctgttcc ttggacgaatgcacaagacgtgctacttcctgggatccgacatggaagcggaggaggacc catcgccctgtgcgtcttcgggatcagggcgtgcgtgcacgctgaaccagactgagtgcc gcgggcgctggccagggcccaatggaggcatcaccaactttgacaacttcttcttcgcca tgctgacagtcttccagtgtgtcaccatggaaggctggaccgatgtgctctactggatgc aagatgccatggggtatgaactgccctgggtgtactttgtgagccttgtcatctttgggt ccttcttcgtcctcaaccttgtgcttggcgtcctgagtggggagttctccaaggagagag agaaagcgaaagctcgcggggacttccagaagcagcgggagaagcagcagatggaggaag acctgcggggctacctggactggatcactcaagccgaagagctggacatggaggacccct ccgccgatgacaaccttggttctatggctgaagagggccgggcgggccatcggccacagc tggccgagctgaccaataggaggcgtggacgtctgcgctggttcagtcattctactcgct ccacacactccaccagcagccatgccagcctcccagccagtgacaccggttccatgacag agacccaaggcgatgaggatgaggaggagggggctctggccagctgtacacgctgcctaa acaagatcatgaaaaccagagtctgccgccgcctccgccgagccaaccgggtccttcggg cacgctgccgtcgggcagtgaagtccaatgcctgctactgggctgtgctgttgctcgtct tcctcaacacgttgaccatcgcctctgagcaccacgggcagcctgtgtggctcacccaga tccaggagtatgccaacaaagtgttgctctgtctgttcacggtggagatgcttctcaaat tgtacggtctgggcccctctgcctatgtgtcttccttcttcaaccgctttgactgctttg tggtctgtgggggcatcctagagaccaccttggtggaggtgggtgccatgcagcccttgg gcatctcagtgctccgatgtgtgcgcctcctcaggatctttaaggtcaccagacactggg cttctctgagcaatctggtggcatccctgctcaattcaatgaaatccatcgcatccttgc tgcttctcctcttcctcttcatcattatcttctccctgcttggcatgcagctgtttgggg gcaagttcaactttgaccagacccacaccaagcgaagcacctttgacacgttcccccagg ccctcctcactgtctttcagatcctgacaggtgaggactggaacgtggtcatgtatgatg gtatcatggcatatggtggccccttcttcccaggaatgttggtgtgcatctatttcatca ttctcttcatctgtggcaactacatcctgttgaacgtgtttcttgccattgctgtggaca acctggccagtggagatgcaggcactgccaaggacaagggcggggagaagagcaatgaga aggatctcccacaggagaatgaaggcctggtgcctggtgtggagaaagaggaagaggagg gtgcaaggagggaaggagcagacatggaggaggaggaggaggaggaagaagaggaagaag aggaagaagaggaagagggtgcagggggtgtggaactcctgcaggaagttgtacccaagg agaaggtggtacccatccctgagggcagcgccttcttctgcctcagccaaaccaacccgc tgaggaagggctgccacaccctcatccaccatcatgtcttcaccaatcttatcctggtgt tcatcatcctcagcagtgtgtccctggccgctgaggaccccatccgagcccactccttcc gcaaccatattctgggttacttcgattatgccttcacctccattttcactgtggagattc tactaaagatgacagtgtttggggccttcctgcaccgcggctccttctgccgtagctggt ttaatatgttggatctgctggtggtcagtgtgtccctcatctcctttggcatccactcca gcgccatctcggtggtgaagattctgcgagtactccgagtactgcggcccctccgagcca tcaacagggccaagggactcaagcatgtggtgcagtgtgtatttgtggccatccggacca tcggaaacatcatgattgtcaccacacttctgcaatttatgttcgcctgcatcggggtgc agctcttcaaggggaaattctacacctgcacggacgaggccaaacacacccctcaagaat gcaagggctccttcctggtatacccagatggagacgtgtcacggcccctggtccgggagc ggctctgggtcaacagtgatttcaactttgacaatgtcctttcagccatgatggccctgt tcactgtctccacctttgaaggctggcctgcactgctatacaaggccatcgatgcatatg cagaggaccacggccccatctataattaccgtgtggagatctcagtgttcttcattgtct acatcatcatcattgcgttcttcatgatgaacatcttcgtgggcttcgtcatcatcactt tccgtgcccagggcgagcaggagtaccaaaactgtgagctggacaagaaccagcgtcaat gtgtggaatatgccctcaaggcccagccactccgccgttacatccccaagaacccgcatc agtatcgtgtgtgggccactgtgaactctgctgcctttgagtacctgatgttcctgctca tcctgctcaacacagttgccctagccatgcagcactatgagcagactgctcccttcaact atgccatggacatcctcaacatggtcttcactggcctcttcactattgagatggtgctca aaatcatcgccttcaagcccaagcattacttcactgatgcctggaacacgtttgacgctc ttattgtggtgggcagcatagtggatattgccgtcactgaagtcaataatggtggccacc ttggcgagagctctgaggacagctcccgcatttccattaccttctttcgcctcttccgag ttatgcggctggtcaagcttctcagtaagggtgaagggatccgcacattgctctggacat tcatcaagtccttccaggccttgccctatgtggctcttctcatcgcaatgatattcttca tctatgccgtcattggcatgcagatgttcggcaaggtggctcttcaggatggcacacaga taaaccgaaacaacaacttccagacctttccacaggctgtgctgcttctgttcaggtgtg ccactggtgaggcatggcaggagataatgcttgccagccttcccggaaatcggtgtgatc ctgagtctgacttcggccctggtgaagagtttacctgtggtagcaattttgccatcgcct atttcatcagcttcttcatgctctgtgccttcctgatcataaatctctttgtggctgtga tcatggacaactttgattatctcaccagagattggtccatcctgggcccccatcaccttg atgaattcaagaggatctggtctgaatatgaccctggggccaagggccgcatcaaacact tggatgtggttgccctgctgagacgtatccagccccctctgggatttgggaagctgtgcc cacaccgagtggcctgcaagagacttgtggcaatgaacatgcccctcaactcagatggga cggtgacattcaacgccacactctttgccctggtccggacatccctgaagatcaaaacag aagggaacctggagcaagccaaccaggagctgcggattgtcatcaaaaagatctggaagc ggatgaaacagaagctgctagatgaggtcatccccccaccagacgaggaggaggtcaccg tgggcaaattctacgccacatttctgatccaggactatttccgcaaattccggcggagga aagaaaaagggctactaggcaacgacgccgcccctagcacctcttccgcccttcaggctg gtctgcggagcctgcaggacttgggtcctgagatgcggcaggccctcacctgtgacacag aggaggaggaagaagaggggcaggagggagtggaggaggaagatgaaaaggacttggaaa ctaacaaagccacgatggtctcccagccctcagctcgccggggctccgggatttctgtgt ctctgcctgtcggggacagacttccagattcactctcctttgggcccagtgatgatgaca gggggactcccacctccagtcagcccagtgtgccccaggctggatccaacacccacagga gaggctctggggctctcattttcaccatcccagaagaaggaaattctcagcccaagggaa ccaaagggcaaaacaagcaagatgaggatgaggaagtccctgatcggctttcctacctag atgagcaggcagggactcccccgtgctcagtccttttgccacctcacagagctcagagat acatggatgggcacctggtaccacgccgccgtctgctgccccccacacctgcaggtcgga agccctccttcaccatccagtgtctgcagcgccagggcagttgtgaggatttacccatcc caggcacctatcatcgtgggcgaaattcagggcccaatagggctcagggttcctgggcaa caecacctcagcggggtcggctcctgtatgccccgctgttgttggtggaagagggcgcag cgggggaggggtacctcggcagatccagtggcccactgcgcaccttcacctgtctgcacg tgcctggaacccactcggaccccagccatgggaagaggggcagtgccgacagcttggtgg aggctgtgcttatctcagagggtctgggcctctttgctcgagacccacgtttcgtggccc tggccaagcaggagattgcagatgcgtgtcgcctgacgctggatgagatggacaatgctg ccagtgacctgctggcacagggaaccagctctctctatagcgacgaggagtccatcctct cccgcttcgatgaggaggacttgggagacgagatggcctgcgtccacgccctctgaattc ccacccctccccaactgctcaataaacctcctgccctcccctccccagcaggaggcaggc atggaccaca

Mouse CACNA1 F tagttgggaggactgtgtgcatgatggtccttatatctcctgaggaggatgtcggaatct CaVl .4 gaagtcgggaaagatacaaccccagagcccagtccagccaatgggactggccctggccct gaatgggggctctgtcctgggcctccaactgtggggactgataccagcggggcgtcaggc ctggggaccccaagaagaaggacccagcacaacaaacacaagactgtggcggtggccagt gctcagagatcacctcgagcgctcttctgcctcacccttactaatcccattcgtcggtcc tgcatcagcattgtagagtggaagccttttgatattctcatcctcctgacaatctttgcc aactgcgtggcattgggggtatatatccccttccctgaggacgactccaacactgctaac cacaacttggaacaggtagaatacgtgttcctggtgattttcaccgtggagacagtgctc aagatcgtagcctatgggctggtgctccatcccagcgcctatattcgcaatggctggaac ctgctcgacttcatcatcgtcgtggtcgggctgttcagcgtgctgctggaacaaggacct gggcggccaggagatgccccgcatactggaggaaagccaggaggcttcgatgtaaaggca ctgcgggcatttagggtgctacgacctctaaggctagtgtctggggtcccgagtctgcac atagtcgtcaattccatcatgaaggcgcttgtgccgctgctgcacattgccctgttggtg ctcttcgtcattatcatttacgccatcatcggactcgagctattcctcggacgaatgcac aagacatgctacttcctgggatctgatatggaagcagaggaggacccatcaccttgtgca tcttctggctctgggcgttcatgcacactgaaccataccgagtgccgcgggcgctggcca ggacccaacggtggcatcacgaacttcgacaattttttctttgccatgctaactgtgttc cagtgtattaccatggaaggctggacagacgtcctctactggatgcaggatgccatgggg tatgagctgccttgggtgtactttgtgagccttgtcatctttgggtccttctttgtcctc aaccttgtgcttggagtcctaagcggggagttctccaaggaaagagaaaaggcaaaagca cgaggtgactttcagaagcttcgggagaagcagcagatggaagaagaccttcggggctac ctggactggatcacacaggctgaggagttagaccttcatgacccctcagtagacggcaac ttggcttctcttgctgaagagggacgggcgggccatcggccacaactgtcagagctgacc aataggaggcgcggacggctgcgatggttcagccactctactcgctccacacactccacc agcagccacgccagcctcccagccagtgacactggctccatgacagacacccctggagat gaggatgaagaagaggggaccatggctagctgtacacgctgcctaaacaagattatgaaa acaaggatctgccgccacttccgccgagccaaccggggtctccgtgcacgctgccgccgg gccgtcaagtccaacgcctgctactgggctgtactgttgctcgtcttcctcaacacgttg accatcgcttcagagcaccatgggcagcctttgtggctcacccagacccaagagtatgcc aacaaagttctgctctgcctcttcactgtggagatgctcctcaaactgtacggcctgggc ccctctgtctacgttgcctcctttttcaaccgctttgactgcttcgtggtctgtgggggc atcctagaaaccactttggtggaggtgggggccatgcagcctcttggcatctcagtgctc cgatgtgtacgtctcctcaggatcttcaaggtcaccaggcactgggcatccctgagcaat ctggtggcatctttgctcaattccatgaagtccatcgcctccttgctgcttctcctcttt ctcttcatcatcatcttctccctgcttggcatgcagctgtttgggggcaagttcaacttt gaccagacccacaccaagaggagcacctttgataccttcccccaagccctcctcactgtc tttcagatcctgactggtgaggattggaacgttgtcatgtatgatggtatcatggcctac ggtgggcccttcttcccagggatgctggtgtgtgtttatttcatcatcctcttcatctgt ggcaactacatcctgctgaacgtgtttcttgccattgccgtggataacctagccagcggg gatgcaggcactgccaaagataagggcagagagaagagcagtgaaggaaaccctccaaag gagaacaaagtattggtgcctggtggagagaatgaggacgcaaagggcgcaagaagtgaa ggagcagcaccaggcatggaggaggaggaggaggaggaggaggaagaagaagaggaggag gaggaggaagaggaaaatggtgcaggacatgtggaactcttgcaggaagtagtacccaag gagaaggtggtacccatccctgaaggcagtgccttcttctgccttagccaaaccaacccg cttcggaaggcctgccacacactcatacatcaccatatcttcaccagtctcatcctagtg ttcatcatcctcagtagtgtgtccctggctgctgaggaccccatccgagctcactccttc cgaaaccatattctgggatattttgattatgccttcacctccatattcactgtggagatt ctactcaagatgacagtgtttggggccttcctgcaccgaggctctttctgccgtagctgg ttcaatctgttggatctccttgtggtcagtgtgtccctcatctccttcggcatccactcc agtgccatctcagttgtgaagattctccgagtcctccgagtcctgcggcctctccgagcc atcaacagagccaagggactcaagcatgtggtgcagtgtgtgttcgtggccatccggacc atcggaaacatcatgattgtcaccaccctcttgcagttcatgttcgcctgcattggtgtt cagctgttcaagggaaaattctacagttgcactgatgaggccaaacacaccctgaaagaa tcgaagggctccttcctcatctaccctgatggagatgtgtcacgacctttggtccgggag cggctctgggtcaacagtgattttaactttgacaacgtcctttcagccatgatggccctg ttcactgtctctacctttgaaggctggcctgegctaetatacaaggccatagatgcaaac gcagaagatgagggccctatctacaattaccatgtggagatatcagtattcttcattgtc tacatcatcatcatcgccttcttcatgatgaacatctttgtgggctttgttatcatcaca ttccgtgcccagggagagcaggagtaccaaaactgtgaactggacaagaaccagcgccag tgtgtggaatatgccctcaaagctcagccactccgccgatacatccctaagaatcctcat cagtaccgcgtgtgggccactgtgaactctcgtgcctttgagtacctcatgtttctgctc atcctgctcaacacggtggccctagccatgcagcactatgaacagactgctccctttaac tatgccatggacatcctcaacatggtcttcactggcctcttcaccattgagatggtgctc aaaatcatcgcctttaaacccaagcattactttgcagatgcctggaatacgtttgatgct ctcattgtagtgggcagtgtagtcgacatcgccgtcacagaagtcaataacggaggccat cttggcgagagttcagaggacacgtcccgcatatctatcacgttctttcgcctcttccga gtcatgaggctggtcaagcttctgagtaagggtgaagggatccgcacactgctctggaca ttcatcaagtctttccaggccttgccctatgtggcacttctcatagcaatgatattcttc atctatgcagtcattggcatgcagatgtttggcttggtggctcttcaggacggcacgcag ataaatcgaaacaacaatttccagacctttccgcaggctgtgctgcttctgttcaggtgt gccactggtgaggcctggcaagagataatgctagccagccttccaggaaatcgatgtgac cctgagtctgactttggcccaggcgaggaatttacctgtggtagcagttttgccatcgtc tacttcatcagcttctttatgctctgtgccttcctgattataaatctctttgtggctgta atcatggataactttgattacctaaccagagattggtctatcctgggaccccaccacctt gatgaattcaagaggatctggtctgaatatgaccccggagccaagggccgcatcaagcac ttggatgtggttgccctgctgagacgcatccagcccccattgggatttggaaagctatgc ccacaccgagtggcctgcaagagactcgtggcaatgaatgtgcccctcaactcagatgga acagtgacattcaacgctacactctttgccctggtgcggacatccctgaagatcaagaca gaagggaacctggatcaagccaaccaggagcttcggatggtcatcaaaaagatctggaag cggataaagcagaaattgttggatgaggtcatccctcctcccgatgaggaggaggtcact gtgggaaaattctatgccacattcctgatccaagattatttccgaaaattccggagaagg aaagaaaaggggctactaggaagagaggccccaacaagcacatcctctgccctccaggct ggtctaaggagcctgcaggacttgggtcctgagatccgtcaagccctcacctatgtcact gaggaagaagaggaagaggaagaggcagtgggtcaggaggctgaggaagaggaagctgag aacaacccagaaccatacaaagactccatagactcccagccccaatctcgatggaactct aggatttcggtgtctctacctgttaaggagaaacttccagattctctctcaactgggccg

agtgatgatgatgggctggctcccaactccaggcagcccagtgtgatacaggctggctcc caaccacacaggagaagctctggggttttcatgttcactatcccggaagaaggaagtatt cagctcaagggaactcaagggcaggacaatcagaatgaggaacaggaactccctgactgg actcctgacctggatcgagcaggccgggactccttcgaacccagtccttttaccacctca ctggtccagcaacacgtaaacgggcacatgtcgacgccgacgtttgctgccccccacgcc tgcaggtcggagccctccttcaccatccagtgtctgcaacgcctgggcagttgtgaagat ttacctatcccaggcacctaccatcgtggacggacctcaggaccaagcagggctcagggt tcctgggcagcccctcctcagaagggtcgactgctatatgcccccctgttgttggtggag gaatctacagtgggtgaaggataccttggcaaacttggcggcccactgcgtaccttcacc tgtctgcaagtgcctggagctcatccgaatcccagccaccgcaagaggggcagtgctgac agtttggtggaggctgtgctcatctccgaaggcctaggtctctttgcccaagacccacga tttgtggccctggccaagcaggagattgcagatgcatgtcacctgaccctggatgagatg gacagtgctgccagtgacctgctggcacagagaaccatctccctttacagtgatgaggag tctattctttcccgctttgatgaagaggacctgggagatgagatggcctgtgtccatgcc ctctaaatccttacccctcatctactgctcaataaactccctgcccttccttcccccaga ggaggcaggcatggaccacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

In some embodiments, CaV1.4 is mammalian CaV1.4. In specific embodiments, CaV1.4 is human CaV1.4. In particular embodiments, CaV1.4 is rodent CaV1.4. In certain embodiments, CaV1.4 is mouse CaV1.4. In particular embodiments, CaV1.4 refers to an CaV1.4 polypeptide, as well as subsequences, fragments, variants (including but not limited to variants resulting from alterative splicing), or derivatives thereof. In some embodiments, CaV1.4 is a polypeptide with an amino acid sequence with at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% homology to amino acid sequences of SEQ ID NOs: 1 or 2. In some embodiments, CaV1.4 is a polypeptide encoded by an m NA sequence with at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% homology to the mRNA sequences of SEQ ID NOs: 3 or 4.

Cavl.4 agonists

In certain embodiments, CaV1.4 agonists described herein activate a CaVl channel containing a CaV1.4 alpha subunit. In particular embodiments, the CaVl.4 agonists described herein activate a channel containing a polypeptide of SEQ ID NOs: 1 or 2, or a fragment or epitope thereof. In certain embodiments, the CaV1.4 agonists activate a channel containing an alpha subunit polypeptide encoded by the mRNA encoded by SEQ ID NOs: 3 or 4, or a fragment or splice variant thereof. In some embodiments, CaVl.4 agonist activates a CaVl channel containing an CaVl.4 alpha subunit polypeptide with an amino acid sequence with at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% homology to amino acid sequences of SEQ ID NOs: 1 or 2. In some embodiments, a CaVl.4 agonist activate a channel containing a CaV1.4 alpha subunit polypeptide encoded by an mRNA sequence with at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% homology to the m NA sequences of SEQ ID NOs: 3 or 4.

Certain embodiments relate to CaV1.4 agonists which activate an L-type voltage-gated calcium channel containing a CaV1.4 alpha subunit. "Activate" as used herein, results in a increase of channel activity. "Activity," as used herein, of the CaV1.4 channel refers to the calcium conductance of the channel. The CaV1.4 channel is considered to be in a closed conformation at resting membrane conditions. "Closed" refers to a conformation of the channel where there is little or no calcium conductance. When the plasma membrane becomes depolarized, the channel adopts an open conformation which allows for calcium conductance. Following the membrane depolarization, the channel remains open for a time before reverting back to a closed conformation. "Open" refers to a conformation where calcium ions are permitted to pass through the channel. A CaV1.4 channel can have one or more open and closed conformations. In some embodiments, a CaV1.4 agonist contacts a CaV1.4 channel and increases its activity. Modification of CaV1.4 channel activity may be achieved by, for example but not limited to, changing the probability that the channel will be in an open or closed conformation, changing the conditions, such as the degree of membrane depolarization, that changes the conformation of the channel, changing the duration of time that the calcium channel remains in an open or closed state, changing the calcium conductance of the channel when it is in an open or closed state, or any combination thereof. In some embodiments, increasing activity of CaV1.4 channel is achieved by converting the channel into an open conformation, increasing the probability that the channel will adopt an open conformation in response to membrane depolarization, reducing the degree of membrane depolarization required to shift the channel into an open conformation, increasing the duration of time the channel remains in an open conformation following depolarization of the plasma membrane, increasing the calcium conductance of the channel when it is in an open conformation, or any combination thereof.

In certain embodiments, CaV1.4 agonist increases CaV1.4 activity. In particular embodiments, increasing activity of a CaV1.4 channel increases the activity by a statistically significant amount. In particular embodiments, increasing activity of CaV1.4 channel results in an increase in channel activity of about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% (including all integers and ranges in between). In certain

embodiments, increasing channel activity results in about a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200- fold, 300-fold, 400-fold, 500-fold, 1,000-fold, 10,000-fold, or greater than 10,000-fold increase (including all integers and ranges in between) of channel activity. Determination if an increase of activity is statistically significant can be made by employing standard methods in the art. A statistically significant increase can refer to CaV1.4 activity in the presence of an agonist compared to a reference standard. Reference standards may include, but are not limited to, CaV1.4 activity before the CaV1.4 channel is contacted with the agonist, a CaV1.4 channel that is contacted with a vehicle control or a control agent that is structurally similar to the CaV1.4 agonist but lacks biological activity, or a reference standard generated by any other methods commonly employed in the art.

In certain embodiments, an increase of activity of a CaV1.4 channel is quantified by standard techniques known in the art. In some embodiments, increase of activity of CaV1.4 channel is measured by contacting the channel with an agonist, measuring the activity of the channel, and comparing the measurement to a measurement of a control channel. In particular embodiments, activity of the channel is assessed by measuring calcium conductance of the channel. Standard techniques for measuring calcium conductance of an L-type voltage-gated calcium channel are known in the art and include, but are not limited to electrophysiological techniques such as patch clamp recording, single channel recording, and whole cell recording; calcium imaging techniques utilizing chemical indicators such as fura-2, indo-1, fluo-3, fluo-4, Calcium Green-1, or genetically encoded indicators such as Pericams, Cameleons, and GCaMP, and measurement of events correlated to L-type voltage-gated channel activity such as expression, phosphorylation, or translocation of a protein.

In certain embodiments, the invention is directed to a CaV1.4 agonist that increases CaV1.4 expression. Expression refers to the level or amount of functional CaV1.4 in a cell, tissue, or organism. An increase in the expression of Cavl.4 can be achieved, for example but not limited to, stimulating transcription of the CACNA1F gene, increasing the amount of CACNA1F mRNA, enhancing the transcription of CACNA1F, increasing the stability of CACNA1F mRNA, increasing the stability of the CaV1.4 polypeptide, increasing the presence of the CaV1.4 channel at the cellular membrane, increasing the stability of the CaV1.4 channel subunits, or any other manipulation that results in an increased amount of functional CaV1.4 channel. In particular embodiments, a CaV1.4 agonist results in an increase in the amount of functional CaV1.4 channels by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%

(including all integers and ranges in between). In certain embodiments, a CaV1.4 agonist increases the amount of functional CaV1.4 channels by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300- fold, 400-fold, 500-fold, 1,000-fold, 10,000-fold, or greater than 10,000-fold increase (including all integers and ranges in between) of channel activity. In particular embodiments, the invention is drawn to a CaV1.4 agonist that increases the amount of functional CaV1.4 channels by a statistically significant amount.

CaVl channel subtypes, including CaV1.4. are expressed in different cell types and tissue types throughout the body, and can be expressed as different variants, including variants that result from alternate splicing of message NA, or different post translational modifications, such as glycosylation or phosphorylation. Different variants of the subtype can be expressed in different tissue or cell types, or alternatively, different variants of the subtype can be expressed in the same tissue or cell type, including in the same cell. In particular embodiments, a CaV1.4 agonist specifically activates all variants of the CaV1.4 subtype. In some embodiments, the CaV1.4 agonist specifically activates a subset of the variants of CaV1.4. In particular embodiments, the CaV1.4 agonist specifically activates CaV1.4 expressed in any cell or tissue. In some embodiments, the CaV1.4 agonist specifically activates CaV1.4 in a subset of cells or tissues of which the subtype is expressed. In particular embodiments, the CaV1.4 agonist activates CaV1.4 channels in B-lymphocytes. In particular embodiments, a CaV1.4 agonist increases the expression of all variants of CaV1.4 channel. In some embodiments, CaV1.4 agonist increases the expression of a subset of the variants of CaV1.4. In particular embodiments, the CaV1.4 agonist increases the expression of functional CaV1.4 expressed in any cell or tissue. In some embodiments, the CaV1.4 agonist specifically increases the expression of CaV1.4 in a subset of cells or tissues that normally express CaV1.4. In particular embodiments, the CaV1.4 agonist increases the expression of functional CaV1.4 channels in B-lymphocytes.

In particular embodiments, a CaV1.4 agonist is administered to a subject. In certain

embodiments, the CaV1.4 agonist is administered to bone marrow. In some embodiments, the CaV1.4 agonist contacts a B-lymphocyte. In some embodiments, the CaV1.4 agonist is a natural or chemically modified polypeptide, an antibody, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified

mononucleotide, a lipopeptide, an antimicrobial, a small molecule, or a pharmaceutical molecule.

In some embodiments, a CaV1.4 agonist is administered to a subject in need thereof. In some embodiments, a CaV1.4 agonist is administered to a subject orally, intravenously, sublingually, buccally, entericly, topically, rectally, subcutaneously, nasally, intraosseously (i.e. by intraosseous infusion), intraperitoneally, intrathecally, transdermally, or transmucosally. In some embodiments, a CaV1.4 agonist is administered to a subject in need thereof once, twice, three times, four times, five times, six times, or more that six times daily. In some embodiments, a CaV1.4 agonist is administered to a subject in need thereof daily, every 48 hours, every 72 hours, every four days, every five days, or every six days. In some embodiments, a CaV1.4 agoinist is administered to a subject in need thereof once every week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, or once over a period of time greater than three months.

Methods of Use

Embodiments include methods relating to the use of Cavl.4 agonists. Particular embodiments are directed to methods of stimulating receptor editing in B-lymphocytes by contacting B-lymphocytes with a CaV1.4 agonist. Receptor editing is a tolerogenic process that attempts to alter the specificity of self recognizing BCRs in immature B-lymphocytes and is the most common method of removing self- reactive B-lymphocytes. Some embodiments contemplate a model whereby receptor editing is a process that reduces the amount of self-reactive B-lymphocytes and increases the amount of circulating mature B-lymphocytes in an organism that are not self-reactive or anergic. In this model, stimulation of receptor editing activity is therefore beneficial for the treatment of a disorder associated with the immune system, or any disorder where reduced levels of auto-reactive B-lymphocytes and/or increased levels of circulating mature B-lymphocytes would benefit a subject.

Certain embodiments are directed to methods of stimulating receptor editing by contacting a B- lymphocyte with a CaV1.4 agonist. In particular embodiments, stimulating receptor editing refers to increasing the probability that a self-reactive B-lymphocyte will undergo receptor editing. In certain embodiments, a CaV1.4 agonist contacts a B-lymphocyte and increases the probability that the B- lymphocyte will under go receptor editing by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,

15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% (including all integers and ranges in between). In some embodiments, a CaV1.4 agonist contacts a B-lymphocyte and increases the probability that the B-lymphocyte will undergo receptor editing by about a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, 10,000-fold, or greater than 10,000-fold (including all integers and ranges in between) increase in probability. Some embodiments are directed to methods of stimulating receptor editing in a population of B-lymphocytes by contacting the population of B-lymphocytes and with a CaV1.4 agonist. In some embodiments, a CaV1.4 agonist contacts a population of B-lymphocytes and increases the amount of B- lymphocytes that undergo receptor editing by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% (including all integers and ranges in between). In some embodiments, a CaV1.4 agonist contacts a population of B- lymphocytes and increases the amount of B-lymphocytes that undergo receptor editing by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70- fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, 10,000-fold, or greater than 10,000-fold (including all integers and ranges in between).

In some embodiments, a CaV1.4 agonist contacts a B-lymphocyte and stimulates receptor editing that modifies sequences of light chain V and/or J genes. In particular embodiments, the CaV1.4 agonist stimulates receptor editing in B-lymphocytes that express an autoreactive BC . In certain embodiments, a Cavl.4 agonist contacts a B-lymphocyte and simulates receptor editing that replaces Ig kappa light chain with Ig lambda light chain in the BCR. In particular embodiments, a CaV1.4 agonist contacts a population of B-lymphocytes and increases the number of B-lymphocytes that undergo receptor editing that modifies sequences of light chain V and/or J genes. In certain embodiments, a CaV1.4 agonist contacts a population of B-lymphocytes and increases the number of B-lymphocytes that undergo receptor editing that replaces Ig kappa light chain with Ig lambda light chain in the BCR.

Particular embodiments are directed to methods of ameliorating cell death by contacting B- lymphocytes with a CaV1.4 agonist. Cell death is an event whereby a cell permanently ceases to perform its functions. Cell death results from the natural process of old cells being replaced by new ones, or from factors such as disease, injury, or death of the organism. Types of cell death include programed cell death which is mediated by an intracellular active, regulated process and includes apoptosis (also known as Type I cell death) and authophagic cell death (also known as

macroauthophagic cell death or Type II cell death). Apoptosis is the process of programmed cell death whereby biochemical events lead to characteristic morphological changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Autophagic cell death is a catabolic process that results in the autophagosomic- lysosomal degradation of bulk cytoplasmic contents, abnormal protein aggregates, and excess or damaged organelles. In particular embodiments, a Cavl.4 agonist contacts a cell and ameliorates cell death. In particular embodiments, the cell is a B-lymphocyte. Ameliorating cell death refers to reducing the probability or likelihood of death in the cell. When referring to more than one cell, for example more than one cell found in a tissue, ameliorating cell death can be used to mean reducing the amount of cells that undergo cell death. In some embodiments, a Cavl.4 agonist contacts a cell and ameliorates programmed cell death. In some embodiments, an inhibitor of SCL26A11 contacts a cell and ameliorates apoptosis. In some embodiments, an inhibitor of SCL26A11 contacts a cell and prevents autophagic cell death. In some embodiments, a Cavl.4 agonist contacts cells and reduces cell death by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and ranges in between). In certain embodiments, a Cavl.4 agonist contacts cells and reduces programmed cell death by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and ranges in between). In particular embodiments, a Cavl.4 agonist contacts cells and reduces apoptosis by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%

(including all integers and ranges in between). In particular embodiments, the amelioration of cell death is statistically significant.

In some embodiments, a CaVl.4 agonist contacts a B-lymphocyte that expresses Cavl.4 channels and stimulates receptor editing and/or ameliorates apoptosis. In some embodiments, the CaV1.4 agonist contacts a B-lymphocyte that is an immature B-lymphocyte or a transitional B- lymphocyte. In particular embodiments the B-lymphocyte is self reactive. In certain embodiments, the B-lymphocyte expresses a self-reactive BCR. In particular embodiments, a CaV1.4 agonist contacts an auto-reactive immature or transitional B-lymphocyte that expresses CaV1.4 and stimulates receptor editing. In some embodiments, a CaV1.4 agonist contacts an auto-reactive immature or transitional B- lymphocyte that expresses CaV1.4 and ameliorates cell death.

In particular embodiments, a CaV1.4 agonist is administered to bone marrow to increase an amount of immunocompetent B-lymphocytes. Without being bound by theory, certain embodiments contemplate that bone marrow contains developing B-lymphocytes, a subset of which are immature or transitional B-lymphocytes that express an autoreactive BCR. Contacting bone marrow with a CaV1.4 agonist will stimulate receptor editing in the B-lymphocytes with autoreactive BCRs, thereby altering the specificity of the BCR. The stimulation of receptor editing by the administration of CaV1.4 agonist to the bone marrow therefore reduces the amount of autoreactive B-lymphocytes and reduces the amount of B-lymphocytes that undergo apoptosis or become anergic. This increases the pool of immature and transitional B-lymphocytes in the bone marrow that can develop into mature immunocompetent B- lymphocytes. In certain embodiments, a CaV1.4 agonist is administered to bone marrow to increase an amount of immunocompetent B-lymphocytes and reduce the amount of B-lymphocytes that undergo apoptosis or clonal anergy.

Some embodiments are directed to methods for increasing an amount of immunocompetent B- lymphocytes by contacting bone marrow with a Cavl.4 agonist. In some embodiments, a CaV1.4 agonist contacts bone marrow and increases the amount of immunocompetent B-lymphocytes by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% (including all integers and ranges in between). In some embodiments, a CaV1.4 agonist contacts bone marrow and increases the amount of immunocompetent B-lymphocytes by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, 10,000-fold, or greater than 10,000-fold (including all integers and ranges in between).

In particular embodiments, the CaV1.4 agonist is administered to bone marrow that contains B- lymphocytes and stimulates receptor editing in a subset of the B-lymphocytes. In some embodiments, the bone marrow contains developing B-lymphocytes. In particular embodiments, the bone marrow contains B-lymphocytes that express Cavl.4. In certain embodiments, the bone marrow contains immature B-lymphocytes and/or transitional B-lymphocytes. In certain embodiments, the B- lymphocytes express an auto-reactive BC . In particular embodiments, the CaV1.4 agonist is administered to bone marrow that contains B-lymphocytes and stimulates receptor editing in a subset of B-lymphocytes that are immature and/or transitional self-reactive B-lymphocytes that express CaV1.4. In some embodiments, the CaV1.4 agonist reduces the number of B-lymphocytes that undergo apoptosis or become anergic. In certain embodiments, the Cavl.4 agonist reduces the amount of B- lymphocytes that leave the bone marrow and express an autoreactive BCR in the subset of B- lymphocytes in the bone marrow.

In particular embodiments, a CaV1.4 agonist is administered to a subject to increase an amount of immunocompetent B-lymphocytes. Without being bound by theory, certain embodiments contemplate that CaV1.4 agonist will stimulate receptor editing in self-reactive B-lymphocytes of the subject. Administering CaV1.4 will therefore reduce the amount of self-reactive B-lymphocytes and increase the pool of B-lymphocytes that can mature into immunocompetent B-lymphocytes. In certain embodiments, the subject is a mammal. In specific embodiments, the subject is a human.

Some embodiments are directed to methods for increasing an amount of immunocompetent B- lymphocytes in a subject by administering CaV1.4 agonist to the subject. In some embodiments, a CaV1.4 agonist is administered to the subject and increases the amount of the subject's

immunocompetent B-lymphocytes by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% (including all integers and ranges in between). In some embodiments, a CaV1.4 agonist contacts bone marrow and increases the amount of immunocompetent B-lymphocytes by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, 10,000-fold, or greater than 10,000-fold (including all integers and ranges in between). In some embodiments, the immunocompetent B-lymphocytes are increased by a statistically significant amount.

In certain embodiments, administering a CaV1.4 agonist to a subject reduces the amount of B- lymphocytes that express an autoreactive BC in the subject. In some embodiments, administration of the CaV1.4 agonist reduces the amount of B-lymphocytes that express an autoreactive BCR by a statistically significant amount. In particular emboidments, a CaV1.4 agonist is administered to a subject and reduces the amount of self-reactive B-lymphocytes of the subject by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and ranges in between).

In certain embodiments, administering a CaV1.4 agonist to a subject reduces the amount of chronically activated B-lymphocytes in the subject. Particular embodiments contemplate that reducing the amount of B-lymphocytes that undergo receptor editing in a subject will increase the amount of chronically activated B-lymphocytes in the subject, and conversely, that increasing the amount of B- lymphocytes that undergo receptor editing in a subject will decrease the amount of chronically activated B-lymphocytes in the subject. In some embodiments, administration of the CaV1.4 agonist reduces the amount of chronically activated B-lymphocytes in a subject by a statistically significant amount. In particular embodiments, a CaV1.4 agonist is administered to a subject and reduces the amount of chronically activated B-lymphocytes by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and ranges in between). In particular embodiments, administering a CaV1.4 agonist to a subject reduces the amount of anergic B-lymphocytes in the subject. Particular embodiments contemplate that administering CaV1.4 agonist to a subject will increase the amount of B-lymphocytes that undergo receptor editing, which will decrease the amount of chronically activated B-lymphocytes in the subject. In some embodiments, administration of the CaV1.4 agonist reduces the amount of anergic B-lymphocytes in a subject by a statistically significant amount. In particular embodiments, a CaV1.4 agonist is administered to a subject and reduces the amount of anergic B-lymphocytes in the subject by about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and ranges in between).

Certain embodiments are directed to methods for treating autoimmune disease by

administering a CaV1.4 agonist to a subject with an autoimmune disease. The pathologic mechanisms of these diseases are complex and often involve a combination of humoral and cellular immune mechanisms. Particular embodiments contemplate that Autoimmune diseases are caused by a breakdown in self-tolerance leading to subsequent immune responses against self, including the production of autoantibodies and deposition of immunoglobulin in affected tissues. Autoantibodies generated from self-reactive B-lymphocytes form immune complexes that promote complement and Fc- receptor mediated tissue inflammation and destruction. Some embodiments contemplate that stimulation of receptor editing by a CaV1.4 agonist will benefit treatment of autoimmune disease by reducing the number of B-lymphocytes that produce autoantibodies. Certain embodiments contemplate that stimulation of receptor editing by a CaV1.4 agonist will benefit treatment of autoimmune disease by reducing the number of chronically activated B-lymphocytes. In some embodiments, a CaV1.4 agonist is administered to a subject for the treatment of an autoimmune disease, wherein the subject has autoreactive B-lymphocytes.

In particular embodiments, CaV1.4 is administered to a subject for the treatment of an autoimmune disease. Exemplary autoimmune diseases include Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome, AutoimmuneAddison's Disease, Autoimmune Hemolytic Anemia,

Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic Inflammatory Demyelinating

Polyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn's Disease, Discoid Lupus, Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease, Guillain-Barre, Hashimoto's Thyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, Insulin dependent Diabetes, Juvenile Arthritis, Lichen Planus, Lupus, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes, Polymyalgia heumatica, Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis,

Vasculitis, Vitiligo, Wegener's Granulomatosis, and myasthenia gravis.

In some embodiments, a CaVl.4 agonist is administered to a subject producing autoantibodies. In particular embodiments, a CaV1.4 agonist is administered to a subject for the treatment of an autoimmune disease that is associated with the production of autoantibodies by B-lymphocytes.

Exemplary autoimmune diseases associated with the production of autoantibodies include Addison's disease, Celiac Disease, Dermatomyositis, Grave's Disease, Hashimoto's Thyroiditis, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia, Reactive Arthritis, Rheumatoid Arthritis, Sjogren Syndrome, Systemic Lupus Erythematosus, and Type I Diabetes.

In certain embodiments, a CaV1.4 agonist is beneficial for the treatment of immunodeficiency.

Immunodeficiency (or immune deficiency) is a state in which the immune system's ability to fight infections disease is compromised or absent. Immunodeficiency disorders are categorized as either congenital (primary) or acquired (secondary). Distinction between primary versus secondary immunodeficiencies are based on whether the cause originates in the immune system itself or is a result of external factors. Secondary immunodeficiency disorders are thought to be more prevalent than primary immunodefiencies, and can be caused by certain drugs or can result from a long-lasting disease or disorder, such as cancer or AIDS. Transplant patients take medications to suppress their immune system as an anti-rejection measure, as do some patients suffering from an over-active immune system. A person who has an immunodeficiency of any kind is said to be immunocompromised. An

immunocompromised person may be particularly vulnerable to opportunistic infections, in addition to normal infections that can effect the general population.

In particular embodiments, a CaV1.4 agonist is administered to a subject for the treatment of immunodeficiency. Certain embodiments contemplate that administration of a CaV1.4 agonist to a subject with immunodeficiency will increase receptor editing in self-reactive immature or transitional B- lymphocytes which will increase the pool of developing B-lymphocytes that can mature into

immunocompetent B-lymphocytes. This will increase the number of mature immunocompetent B- lymphocytes in the subject, thereby treating the subject's immunodeficiency. In particular embodiments, a CaVl.4 agonist is administered to a subject for the treatment of a primary immunodeficiency. In certain embodiments, the CaV1.4 agonist is administered for a primary immunodeficiency where the immunodeficiency is characterized, at least in part, by a decreased number of or a dysfunction in B-lymphocytes. Examples of primary immunodeficiency disorders involving decreases or dysfunctional B-lymphocytes include, but are not limited to, Severe combined immunodeficiency (SCID), RAG 1/2 deficiency, DCLRE1C deficiency, adenosine deaminase (ADA), deficiency, reticular dysgenesis, Omenn syndrome, DNA ligase type IV deficiency, Cernunnos deficiency, CD40 ligand deficiency, CD40 deficiency, Purine nucleoside phosphorylase (PNP) deficiency, CD3y deficiency, CD8 deficiency, ZAP-70 deficiency, Calcium channel deficiency, MHC class I deficiency, MHC class II deficiency, Winged helix deficiency, CD25 deficiency, STAT5b deficiency, Itk deficiency, DOCK8 deficiency, Activated PI3K Delta Syndrome, alymphocytosis, Glanzmann-Riniker Syndrome, Severe Mixed Immunodeficiency Syndrome, Thymic Alymphoplasia, X-linked severe combined immunodeficiency, Adenosine Deaminase Deficiency, Purine Nucleoside Phosphorylase Deficiency, Reticular Dysgenesis, Omenn Syndrome, and Bare Lymphocyte Syndrome. In particular embodiments, the subject has a primary immunodeficiency characterized, at least in part, by primary antibody deficiencies. In primary antibody deficiencies, one or more isotypes of immunoglobulin are decreased or don't function properly. Primary immunodeficiencies characterized by primary antibody deficiencies include X-linked agammaglobulinemia (btk deficiency, or Bruton's agammaglobulinemia), μ-Heavy chain deficiency, I 5 deficiency, Iga deficiency, BLNK deficiency, thymoma with immunodeficiency, common variable immunodeficiency (CVID), ICOS deficiency, CD19 deficiency, TACI (TNFRSF13B) deficiency, BAFF receptor deficiency, Hyper-lgM syndrome, heavy chain deletions, kappy chain deficiency, isolated IgG subclass deficiency, selective immunoglobulin A deficiency, and Transient hypogammaglobulinemia of infancy (THI). In some embodiments, a CaV1.4 agonist is administered to a subject with a primary

immunodeficiency, thereby treating the primary immunodeficiency in the subject.

In particular embodiments, a CaV1.4 agonist is administered to a subject for the treatment of a secondary immunodeficiency. In certain embodiments, the CaV1.4 agonist is administered for a secondary immunodeficiency where the immunodeficiency is characterized, at least in part, by a decreased number of or a dysfunction in B-lymphocytes. Secondary immunodeficiencies can result from various immunosuppressive agents, for example, malnutrition, aging, and particular medications (e.g. chemotherapy, disease-modifying antirheumatic drugs, immunosuppressive drugs after organ transplants, glucocorticoids). Many specific diseases directly or indirectly cause immunosuppression. This includes many types of cancer, particularly those of the bone marrow and blood cells (leukemia, lymphoma, multiple myeloma), and certain chronic infections. Immunodeficiency is also the hallmark of acquired immunodeficiency syndrome (AI DS), caused by the human immunodeficiency virus (HIV).

In certain embodiments, a CaVl.4 agonist is administered to a subject for the treatment of a secondary immunodeficiency, wherein the secondary immunodeficiency is caused, at least in part, by cancer chemotherapy treatment, glucocorticoid therapy, or by immunosuppressive treatments following organ transplantation, marrow transplantation, tissue transplantation, or glucocorticoid therapy. In particular embodiments, CaV1.4 is administered to a subject for the treatment of an immunodeficiency caused by a disease, for example but not limited to Multiple Myeloma, Chronic Lymphoid Leukemia, AIDS, Lymphoma, or Chronic Granulomatous Disease.

In particular embodiments, a CaV1.4 agonist is administered to a subject with immunodeficiency having or at risk of having an opportunistic infection. An opportunistic infection is an infection caused by bacterial, viral, fungal, or protozoan pathogens that take advantage of a host with a weakened immune system. Many of these pathogens do not cause disease in a healthy host that has a normal immune system. A compromised immune system, however, presents an "opportunity" for the pathogen to infect. Examples of opportunistic infections include, but are not limited to, Acinetobacter baumanni, Aspergillus sp., Candida albicans, Clostridium difficile, Cryptococcus neoformans, Cryptosporidium sp., Cytomegalovirus, Geomyces destructans, Giardia intestinalis, Histoplasma capsulatum, Hemophilus influenza, Isospora belli, Polyomavirus JC polyomavirus, Kaposi's Sarcoma caused by Human herpesvirus 8 (H HV8), Kaposi's sarcoma-associated herpesvirus (KSHV), Legionnaires' Disease (Legionella pneumophila), Microsporidium, Mycobacterium avium complex (MAC) (Nontuberculosis

Mycobacterium), Pneumocystis jirovecii, Pseudomonas aeruginosa, Staphylococcus aureus,

Streptococcus pneumonia, Streptococcus pyogenes, or Toxoplasma gondii.

EXAMPLES EXPERIMENTAL PROCEDURES

Mice: CaV1.4 knock out (Cacnalf¹ ) mice that have been previously described (Mansergh, F. et al. Hum Mol Genet 14, 3035-3046 (2005)) were bred onto C57BL/6J (B6) background for at least 13 generations. All studies followed guidelines set by both the University of British Columbia's Animal Care Committee and the Canadian Council on Animal Care.

Splenic B-Lymphocyte purification: B lymphocytes from the spleen of mice were first purified by negative selection using the EasySep™ Mouse B Cell Isolation Kit from StemCell (Vancouver, Canada). The isolated cells were then further enriched for follicular B lymphocytes bypositively selecting for CD23 using magnetic bead cell separation technique using MACS CD23 MicroBeads (Miltenyi Biotec). The purity of the isolated follicular B lymphocytes was generally >85%.

Flow Cytometry: Freshly isolated splenocytes or cultured follicular B lymphocytes were stained with commercially available antibodies against B220, CD21, CD23, CD86, MHCII, and 7AAD for 20 min in PBS at 4°C in the dark. Cells were then washed and analyzed using a BDTM LSR II flow cytometer (BD Biosciences) with FACSDivaTM software and analyzed with FlowJo software (Treestar).

Activation assay: Purified follicular B lymphocytes were cultured in RPMI supplemented with 10% FBS, 10 mM L-glutamine, HEPES, 50 μΜ β-mercaptoethanol and Pen/Strep and stimulated with different concentrations of either anti-CD40, anti-IgM, or LPS for 24 hours. The cells were then analyzed using flow cytometry as above.

Ca²⁺ flux measurements: Freshly isolated splenocytes were loaded with the Ca²⁺ dyes Fura-Red and Fluo-4 and also surface-stained with anti-B220, anti-CD21 and anti-CD23. The loaded cells were analyzed by flow cytometry on a BD-LSRII by gating on follicular B lymphocytes and plotting the ratio of Fluo-4/FuraRed over time during which 10 ug/ml of anti-IgM and ionomycin were added to induce Ca²⁺- flux. Fluo-4 was used to quantify basal levels of intracellular calcium before stimulation.

Anti-DNA antibodies detection: The serum of mice was harvested by cardiac puncture and subsequent centrifugation of the collected blood. The anti-DNA antibodies in the serum were detected using ELISA plates coated with salmon-sperm DNA.

Albuminuria quantification in urine: Urine of mice was collected and Albustix Strips from Siemens were used to quantify albumin (protein) concentration. The possible approximated results were negative, trace, 30, 100, 300 or > 2000 mg/dl albumin.

Cytokine detection: Purified follicular B lymphocytes were cultured in RPMI (Invitrogen) supplemented with 10% FBS, 10 mM L-glutamine, HEPES, 50 μΜ β-mercaptoethanol and Pen/Strep and stimulated with 1 ug/ml of LPS for 24 hours. The supernatants were then analyzed for the presence of different cytokines (IL-6, IL-10, MCP-1, IFN-γ, TNF, and IL-12p70) using the Cytometric Bead Array (CBA) - Mouse Inflammation Kit from BD Biosciences.

Receptor editing assay: Bone marrow of mice was flushed with media followed by lysis of the red blood cells. The purified bone marrow cells were cultured in RPMI supplemented with 10% FBS, 10 mM L-glutamine, HEPES, 50 μΜ β-mercaptoethanol and Pen/Strep and stimulated with different concentrations of anti-IgM (clone R6-60.2 from BD Biosciences) for 72 hours. The cells were then stained for B220 (RA6B2 from AbLab at the University of British Columbia), CD93 , IgD, IgK and IgL and analyzed by flow cytometry. Immature/transitional B lymphocytes were identified as B220+ CD93- IgDint. Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of that disclosure or the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

EXAMPLE 1

CAVI.4 KG DEFICIENT MICE HAVED ALTERED PERIPHERAL B-LYMPHOCYTE DEVELOPMENT

B-lymphocytes are produced in the bone marrow and migrate to the spleen and other secondary lymphoid tissues as they mature and differentiate into immunocompetent B-lymphocytes. Experiments were performed to examine B-lymphocyte development in CaVl.4 knockout [Cacnalf^1'). To determine levels of B-lymphocytes present in bone marrow, bone marrow was collected from wild- type and Cacnalf^1' mice and cells were dissociated and labeled for flow cytometry analysis. Cells labeled positive for B220 (B220+) were identified as B-lymphocytes. No difference was observed in the percentage or total number of B220+ cells, indicating that Cacnalf^1' and wild-type mice have similar numbers of B-lymphocytes present in bone marrow (Figure la). Next, cells from bone marrow of wild- type and Cacnalf^1' mice were examined by flow cytometry analysis to determine the presence of B- lymphocytes at different developmental stages. Different developmental stages of the labeled cells were identified by according to the Hardy gating scheme: pre-pro B-lymphocytes (Pre-pro-B),

B220⁺CD43⁺BP-1^~HAS^~; pro B-lymphocytes (pro-B), B220⁺CD43⁺BP-1^~HSA⁺; early pre B-lymphocytes (early pre-B), B220⁺CD43⁺BP-1⁺HSA⁺; late pre B-lymphocytes (late pre-B), B220⁺CD43^"lgM^"lgD^"; immature pre B- lymphocytes (immature), B220⁺CD43^"lgM⁺lgD^"; and recirculating mature B-lymphocytes (mature), B220⁺CD43^"lgM⁺lgD⁺. While no difference was seen between wild-type and Cacnalf^1' mice in Pre-pro-B, pro-B, early pre-B, late pre-B, or immature populations of B-lymphocytes, bone marrow from Cacnalf^1' mice had reduced number of mature B-lymphocytes compared to wild-type bone marrow (Figure lb).

B-lymphocytes were next quantified in the spleen. Spleens from Cacnalf^1' and wild-type mice were collected, and cells were dissociated and labeled for flow cytometry analysis. Reduced numbers and reduced frequency of B220⁺ cells were observed in Cacnalf^1' spleen as compared to wild-type spleen (Figure lc). Number of cells in different B-lymphocyte populations were then examined. B- lymphocyte populations found in spleen were defined as according to Allman and Pillai: transitional Tl, CD93⁺CD23 lgM^highlgD^7l0WCD21/357^l0W; transitional T2, CD93⁺CD23lgM^highlgD^highCD21/35^low; transitional T3, CD93⁺CD23⁺lgM^l0WlgD^highCD21/35^l0W; follicular type I (Fol), CD93 CD23⁺lgM^lowlgD^highCD21/35^mid; follicular type II (Fo2), CD93^"/lowCD23⁺lgM^highlgD^highCD21/35^mid; marginal zone precursor (MZP) CD93^" /iow_CD23+s|gMhigh_CDld+|gDhigh_{CD21 /35}high. _{and margina}| _{zone ( MZ)} CD93 CD23 lgM^highlgD^l0WCD21/35^high.

For each subset of B-lymphocytes examined, spleens from Cacnalf ^f~ mice had reduced number of cells compared to wild-type spleen (Figure Id). B-lymphocytes were also collected from the peritoneal cavity. The percentage of cells from the peritoneal cavity that were identified as B-lymphocytes was reduced in Cacnalf^' mice as compared to wild-type mice (Figure le). B-lymphocyte populations present in the peritoneal cavity were also measured. B-lymphocyte populations were defined as follows: conventional B2 B-lymphocytes, B220⁺CDllb^"; Bla B-lymphocytes, B220⁺CDllb⁺CD5⁺; and Bib B-lymphocytes, B220⁺CDllb⁺CD5^". Cell numbers of these populations were reduced in Cacnalf ^f~ mice as compared to wild-type mice (Figure If). These data are consistent with the notion B-lymphocytes from Cacnalf^ mice show an impaired developmental transition from immature to mature stages.

Experiments were performed to determine if the CaVl.4 channels expressed on B-lymphocytes contribute to B-lymphocyte development. Congenic CD45.1+ wild-type recipient mice were lethally irradiated to reduce B-lymphocyte levels and then were intravenously injected with a 1:1 mixture of wild-type CD45.1+CD45.2+ (competitor) plus wild-type CD45.2+ (donor) bone marrow or wild-type CD45.1+CD45.2+ (competitor) plus Cacna If ^_CD45.2+ (donor). Eight weeks after injection, cells were collected from bone marrow (Figure 2a), spleen (Figure 2b), and peritoneal cavity (Figure 2c), labeled and analyzed with flow cytometry. B-lymphocyte populations were identified as described above. For all cell populations, lower ratios of donor B-lymphocytes to competitor B-lymphocytes were observed for Cacnalf^' donor lymphocytes as compared to wild-type donor lymphocytes. These results show that a cell-intrinsic CaV1.4 function is required for normal B cell development.

EXAMPLE 2

CAVI.4 CONTRIBUTES TO CALCIUM SIGNALING IN B-LYMPHOCYTES

Freshly isolated splenocytes were stained with antibodies to identify follicular B-lymphocytes. B220+ CD21+ CD23+ splenocytes were identified as follicular B-lymphocytes and isolated with flow cytometry. Internal Ca²⁺ concentrations were measured in follicular B lymphocytes from Cacnalf^ and wild-type mice. Follicular B-lymphocytes and were loaded with the Ca²⁺ sensitive dyes Fluo-4 and Fura- Red. The Cacnalf ^f~ B-lymphocytes showed an impaired mobilization of Ca²⁺ after stimulation by adding ct-lgM and subsequently ionomycin to the cell culture media (Figure 3a). Next, EGTA, a Ca²⁺ chelator, was added to the media to test the Ca²⁺ mobilization of splenocytes in response to ct-lgM in the absence of extracellular Ca²⁺. Stimulation by ct-lgM in the presence of EGTA transiently increased intracellular Ca²⁺ levels in splenocytes, although the increase was impaired in Cacnalf^' splenocytes. When extracellular Ca²⁺ levels were restored, intracellular calcium levels of splenocytes increased in the presence of ct-lgM; however, this increase was impaired in Cacnalf ^f~ cells (Figure 3b). Cacnalf^' and wild-type Spenocytes were then tested with thapsigargin (Tg). Thapsigargin raises intracellular (cytosolic) Ca²⁺ concentration by blocking the ability of the cell to pump calcium into the sarcoplasmic and endoplasmic reticula which causes these stores to become depleted. Store-depletion can secondarily activate plasma membrane calcium channels, allowing an influx of calcium into the cytosol. When splenic B-cells were stimulated with thapsigargin, Cacnalf^1' splenocytes showed impaired Ca2+ influx compared to wild-type splenocytes (Figure 3c). Following the addition of EGTA to the cell media, intracellular Ca2+ levels returned to baseline, indicating that the increased internal Ca2+ response to thapsigargin was due to import of extracellular calcium (Figure 3c).

In resting conditions, Cacnalf^1' B-lymphocytes had elevated basal internal Ca²⁺ concentrations as compared to wild-type B-lymphocytes (Figure 3d). Of note, a similar phenotype was observed in previously activated resting wild-type B lymphocytes as compared to resting non-activated wild-type B lymphocytes (data not shown). A similar phenomenon has been previously observed in tolerant B- lymphocytes as compared to naive B-lymphocytes (Healy, et al. Immunity 6, 419-428 (1997)), indicating that by analogy, the Cacnalf^ B-lymphocytes might be rendered anergic during their development.

Calcium channel activity was then examined in splenic B-lymphocytes using whole cell voltage clamp techniques. Cells were depolarized by 500 ms step pulse to +10 mV from a holding potential of -80 mV in the presence of extracellular barium. BCRs of wild-type and Cacnalf^' splenic B-lymphocytes were stimulated with ct-lgM (wild-type, n = 7; Cacnalf^, n = 6) and inward barium currents were detected in wild-type, but not Cacnalf^' cells. This inward current was blocked following the application of 4 μΜ of Bay K8644, a CaV channel inhibitor (Figure 4a). Next, current density at +10 mV was compared between wild-type (n=7) and Cacnalf^' (n=6) splenic B-lymphocytes. Current values were normalized to capacitance values for each cell. Cacnalf ^f~ cells had a smaller current density compared to wild-type splenocyte B-lymphocytes (Figure 4b). Current was stimulated in wild-type splenic B- lymphocytes and inward barium currents were measured from wild-type splenic B-lymphocytes (n = 4) before and after treatment with 10 μΜ of nifedipine, a CaV channel inhibitor. Nifedipine drastically reduced the inward barium current (Figure 4c). Next, current-voltage relationship of relationship of wild-type B-lymphocyte inward current recorded with normal (Figure 4d) or modified (+ Ba²⁺)

Tetraethylammonium (TEA) internal (Figure 4e) solutions in the presence of 4 μΜ of Bay K8644 in the external solution with a ramp pulse protocol following BC activation. The ramp pulse protocol spans the range of -130 to +70 mV over 200 ms from a holding potential of -80 mV. Modified Boltzmann equation fits were calculated to fit the l-V relationships. (Figure 4d and 4e). Cells with modified TEA internal solutions (+ Ba²⁺) had reduced reversal potential between compared to cells recorded with normal TEA internal solution (Figure 4f). Taken together, these experiments demonstrate that Ca_v1.4 mediates Ca²⁺ entry across the plasma membrane of splenic B-lymphocytes.

EXAMPLE 3

CAVI.4 KG B-LYMPHOCYTES HAVE ALTERED ACTIVATION STATUS

Activation of follicular B-lymphocytes isolated from spleen was tested by stimulating isolated wild-type and Cacnalf^' B-lymphocytes with ct-lgM. B-lymphocytes were stained for surface activation markers CD69 and CD86 and analyzed by flow cytometry. Cacnalf^' cells were not activated to the same extent as wild-type cells when compared to unstimulated controls (Figure 5a and 5b). B- lymphocytes were then stimulated with ct-lgM in the presence of Carboxyfluorescein succinimidyl ester (CFSE), a fluorescent dye that is internalized by proliferating cells. The ct-lgM -induced proliferation was impaired Cacnalf^1' cells as compared to wild-type cells (Figure 5C). These defects were not observed when using lipopolysaccharide (LPS) as stimulus (Figures 5a-5c), suggesting that the activation deficit of Cacnalf^1' B-lymphocytes is linked to BCR and not other toll-like receptors such as TLR4.

Cacnalf^1' B-lymphocytes were characterized by examining their activation status. Follicular B- lymphocytes were stained with the activation markers CD86 and major histocompatibility complex II (MHCII). Cacnalf^1' B-lymphocytes displayed higher levels of activation markers on their cell surface than wild-type B-lymphocytes. Cacnalf^1' B-lymphocytes were observed as more granular (as measured by the side-scattered light (SSC) plot), which is indicative of a more activated phenotype (Figure 5d). Therefore in the Cacnalf^1' mouse, the B-lymphocytes appear to constitutively exhibit a chronic state of activation.

Wild-type and Cacnalf^1' B-lymphocytes isolated from spleen were examined for B cell activating factor (BAFF) signaling. Surface expression of BAFF receptors was examined with flow cytometry analysis. Surface density of BAFF receptors in total B-lymphocytes and in the B-lymphocyte subset populations Tl B-lymphocytes, transitional B2 B-lymphocytes, follicular B-lymphocytes, and marginal zone B-lymphocytes were reduced in Cacnalf B-lymphocytes as compared to wild-type cells (Figure 6a). Survival of purified splenic B-lymphoctes cultured in the presence of the recombinant mouse BAFF for 72 h were reduced in Cacnalf^1' B-lymphocytes as compared to wild-type cells (Figure 6b).

Cacnalf^1' and wild-type mice were tested for their ability to generate an antibody response. Mice were immunized intraperitoneal^ with TNP-Ficoll, a T cell-independent type-2 antigen. Serum samples were analyzied with ELISA to determine levels of specific antibodies elicited by the

immunization. Anti-lgM (Figure 7a) and anti-lgG3 (Figure 7b) antibody responses were quantified at 0 days and 7 days after the immunization. Cacnalf^1' mice generated impaired antibody responses to TNP-Ficoll as compared to wild-type mice.

EXAMPLE 4

CAVI.4 KG MICE DISPLAY AN AUTOIMMUNE PHENOTYPE

Isolated Cacnal ^7" B-lymphocytes displayed similar phenotypes to tolerant wild-type B- lymphocytes (Example 3). To determine if Cacnalf^1' mice exhibited impaired B-lymphocyte tolerance mechanisms, sera from Cacnalf^1' and wild-type mice were evaluated for the presence of autoantibodies. To this end, sera of young (8 weeks old) and old (40 weeks old) Cacnalf^1' and wild-type mice were collected and analyzed for the presence of anti-DNA antibodies with an anti-DNA ELISA (n = 6 per group). While there was no significant difference in the amount of anti-DNA antibodies between the wild-type - and CaVl.4 Cacnalf^1' sera in young mice, the sera of the old Cacnalf^1' mice contained significantly more anti-DNA antibodies than the sera of the old wild-type controls (Figure 8a). These data demonstrated that older CaV1.4 Cacnalf^1' mice accumulate auto-antibodies, which is a phenotype associated with autoimmune disease.

Another aspect of autoimmune disease is damage to the kidneys due to Ig deposits, which leads to proteinuria in the urine. To assess proteinuria, the levels of albumin in the urine of 70 week-old mice were measured using Albustix Strips (n > 3 per group). The CaV1.4 Cacnalf^1' mice had significantly higher levels of protein compared to wild-type mice, confirming the defects in autoimmunity (Figure 8b). These data support the conclusion that there exists an emergent B-Lymphocyte autoimmunity in the CaV1.4 Cacnalf^1' mice.

Follicular B-lymphocytes were next examined to determine if Cacnalf^1' B-lymphocytes exhibit an inflammatory program in comparison with wild-type controls. Follicular B lymphocytes were stimulated with LPS and cultured for 24 hours and then quantified the levels of pro-inflammatory cytokines in the supernatant. The concentrations of IFN-g, TNF and IL-6 were determined using cytometric beads. Cacnalf B-lymphocytes secreted higher levels of the pro-inflammatory cytokines interferon-gamma (IFN-g), tumor necrosis factor (TNF) and interleukine-6 (IL-6) (Figure 8c), all of which have been implicated in inflammation and autoimmune pathology. EXAMPLE 5

CAVI.4 KG MICE DISPLAY AN AUTOIMMUNE PHENOTYPE

Further experiments were performed to determine if the auto-reactive B-Lymphocyte phenotype of the Cacnalf ^f~ mice was due to a perturbation of B-Lymphocyte receptor editing.

Supporting the hypothesis that Cavl.4 plays a crucial role in this process, RNA-based expression data from the Immunological Genome Project (www.immgen.org) reveals that CaV1.4 is absent during early B-Lymphocyte development but is then induced during the immature/transitional B-Lymphocyte developmental stage (Heng, et ai, Nat Immunol 9, 1091-1094, (2008)), which is when receptor editing takes place. When B-lymphocytes reach maturity, CaV1.4 expression levels decrease.

Cacnalf ^f~ and wild-type immature B-lymphocytes were analyzied for their ability of to induce receptor editing. B220+ cells from the bone marrow of Cacnalf ^f~ and wild-type mice were stimulated with different concentrations of ct-lgM for 20 hours and then labeled for flow cytometry analysis.

Immature/transitional B-Lymphocytes (B220+ CD93- IgDint) were analyized for IgK and IgA light chain expression. Wild-type immature B-lymphocytes stimulated with ct-lgM underwent receptor editing and replaced their IgK with their IgA light chains. This process was grossly impaired in Cacnalf^' immature B- lymphocytes (Figure 9) indicating that these cells are have impaired receptor editing.

EXAMPLE 6

B-LYMPHOCYTE POPULATIONS IN HEL TRANSGENIC CAV1.4 KG MICE

Experiments are performed in B-lymphocytes obtained from Cacnalf ^f~ mice expressing a transgenic BCR against an exogenous antigen {Hel^ta/Cacna If ^_mice). This will demonstrate that the phenotypes observed in the CaV1.4 mice are due to compromised receptor editing as opposed to impaired Ca²⁺ signaling in developing or mature B-lymphocytes. Because these B-lymphocytes lacking CaV1.4 will express a transgenic BCR against the exogenous antigen hel (i.e. a non self-recognizing BCR), these B-lymphocytes will not undergo receptor editing. No differences are observed between wild-type and Hel^ts '/Cacnalf ^'''^"mice with respect to properties of B-lymphocyte populations, B-lymphocyte development, or B-lymphocyte-dependent immune activities.

Claims

Claims:

1. A method for inducing receptor editing in a B-lymphocyte, comprising contacting the B- lymphocyte with a Cavl.4 agonist, thereby inducing receptor editing in the B-lymphocyte.

2. The method of claim 1, wherein the B-lymphocyte expresses Cavl.4.

3. The method of claim 2, wherein the B-lymphocyte is an immature B-lymphocyte or a transitional lymphocyte.

4. The method of any of claims 1-3, wherein the B-lymphocyte expresses an autoreactive B cell receptor (BC ).

5. The method of claim 4, wherein the receptor editing modifies sequences of light chain V and/or J genes.

6. The method of claim 4 or 5, wherein the receptor editing replaces Ig kappa light chain with Ig lambda light chain in the BCR.

7. The method of any of claims 1-6, wherein the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified

8. The method of any of claims 1-7, wherein the Cavl.4 agonist increases calcium conductance of Cavl.4.

9. The method of any of claims 1-8, wherein the Cavl.4 agonist increases Cavl.4 expression in the cell.

10. The method of any of claims 1-9, wherein the Cavl.4 agonist increases membrane localization of Cavl.4 in the cell.

11. A method for ameliorating death in a B-lymphocyte, comprising contacting the cell with a Cavl.4 agonist, thereby ameliorating death in the B-lymphocyte, wherein the B-lymphocyte expresses an autoreactive B cell receptor (BC ).

12. The method of claim 11, wherein the immune cell expresses Cavl.4.

13. The method of claim 12, wherein the B-lymphocyte is an immature B-lymphocyte or a transitional lymphocyte.

14. The method of any of claims 11-13, wherein the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified

15. The method of any of claims 11-14, wherein the Cavl.4 agonist stimulates receptor editing in the cell.

16. The method of any of claims 11-15, wherein the Cavl.4 agonist increases calcium conductance of Cavl.4.

17. The method of any of claims 11-16, wherein the Cavl.4 agonist increases Cavl.4 expression in the cell.

18. The method of any of claims 11-17, wherein the Cavl.4 agonist increases membrane localization of Cavl.4 in the cell.

19. The method of any of claims 11-18, wherein the cell death is apoptosis.

20. A method for increasing an amount of immunocompetent B-lymphocytes, comprising contacting bone marrow with a Cavl.4 agonist, wherein the bone marrow comprises B-lymphocytes, thereby increasing the amount of the immunocompetent B-lymphocytes.

21. The method of claims 20, wherein the B-lymphocytes are immature B-lymphocytes and/or transitional B-lymphocytes.

22. The method of claims 20 or 21, wherein a subset of the B-lymphocytes expresses an autoreactive BC .

23. The method of any of claims 20-22, wherein the subset of the B-lymphocytes express Cavl.4.

24. The method of claim 22 or 23, wherein contacting the bone marrow with the Cavl.4 agonist stimulates receptor editing in the subset of the B-lymphocytes.

25. The method of any of claims 22-24, wherein contacting the bone marrow with the Cavl.4 agonist ameliorates cell death in the subset of the B-lymphocytes.

26. The method of claim 25, wherein the cell death is apoptosis.

27. The method of any of claims 22-26, wherein contacting the bone marrow with the Cavl.4 agonist reduces an amount of transitional B-lymphocytes that leave the bone marrow and express an autoreactive BCR.

28. The method of any of claims 20-27, wherein the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified mononucleotide, a lipopeptide, an antimicrobial, a small molecule, or a pharmaceutical molecule.

29. A method for increasing an amount of immunocompetent B-lymphocytes in a subject, comprising administering to the subject a Cavl.4 agonist, thereby increasing the amount of the immunocompetent B-lymphocytes in the subject.

30. The method of claim 29, wherein the subject is a mammal.

31. The method of claim 30, wherein the subject is a human.

32. The method of any of claims 29-31, wherein administering to the subject the CaVl.4 agonist reduces an amount of chronically activated B-lymphocytes in the subject.

33. The method of any of claims 29-32, wherein administering to the subject the CaV1.4 agonist reduces an amount of anergic B-lymphocytes in the subject.

34. The method of any of claims 29-33, wherein administering to the subject the CaV1.4 agonist reduces an amount of B-lymphocytes that express an autoreactive BC .

35. The method of any of claims 29-34, wherein administering to the subject the CaV1.4 agonist increases an amount of cells that undergo receptor editing, wherein the cells that undergo receptor editing are B-lymphocytes that express an autoreactive BCR.

36. The method of any of claims 29-35, wherein the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified mononucleotide, a lipopeptide, an antimicrobial, a small molecule, or a pharmaceutical molecule.

37. A method for treating an autoimmune disease in a subject, comprising administering to the subject a Cavl.4 agonist.

38. The method of claim 37, wherein the subject is a mammal.

39. The method of claim 38, wherein the subject is a human.

40. The method of any of claims 37-39, wherein the subject produces an autoantibody.

41. The method of claim 40, wherein administering to the subject the Cavl.4 agonist reduces autoantibody in the subject.

42. The methods of any of claims 37-41, wherein the autoimmune disease is Addison's disease, Celiac Disease, Dermatomyositis, Grave's Disease, Hashimoto's Thyroiditis, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia, Reactive Arthritis, Rheumatoid Arthritis, Sjogren Syndrome, Systemic Lupus Erythematosus, Type I Diabetes.

43. The methods of any of claims 37-42, wherein the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified

44. A method for treating immunodeficiency in a subject, comprising administering to the subject an agonist of Cavl.4.

45. The method of 44, wherein the subject is a mammal.

46. The method of 45, wherein the subject is a human.

47. The method of any of claims 44-46, wherein the immunodeficiency is a primary

immunodeficiency.

48. The method of any of claims 44-47, wherein the immunodeficiency is an acquired immunodeficiency.

49. The method of claim 48, wherein the subject has undergone cancer chemotherapy, organ transplantation, marrow transplantation, tissue transplantation, or glucocorticoid therapy.

50. The method of any of claims 44-49, wherein the immunodeficiency is a humoral immune deficiency.

51. The method of claim 50, wherein the subject has Multiple Myeloma, Chronic Lymphoid Leukemia, AIDS, Lymphoma, or Chronic Granulomatous Disease.

52. The method of any of claims 44-51, wherein the subject has or is at risk of having at least one opportunistic infection.

53. The method of any of claims 44-52, wherein the at least one opportunistic infection is selected from the list consisting of Acinetobacter baumanni, Aspergillus sp., Candida albicans, Clostridium difficile, Cryptococcus neoformans, Cryptosporidium sp., Cytomegalovirus, Geomyces destructans, Giardia intestinalis, Histoplasma capsulatum, Hemophilus influenza, Isospora belli, Polyomavirus JC polyomavirus, Kaposi's Sarcoma caused by Human herpesvirus 8 (HHV8), Kaposi's sarcoma-associated herpesvirus (KSHV), Legionnaires' Disease (Legionella pneumophila), Microsporidium, Mycobacterium avium complex (MAC) (Nontuberculosis Mycobacterium), Pneumocystis jirovecii, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, Streptococcus pyogenes, or Toxoplasma gondii.

54. The method of any of claims 44-53, wherein the Cavl.4 agonist is an antibody, a natural or chemically modified polypeptide, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, NAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified