CA2003459A1 - Muscle nicotinic acetylcholine receptors: their characterization preparation and use as diagnostic screening agents - Google Patents

Abstract

Abstract of the Disclosure Disclosed is the basis for novel assays for detecting and pharmacologically characterizing agents that affect human muscle nicotinic acetylcholine receptor function. Also disclosed are recombinant means for producing human muscle nicotinic acetylcholine receptor and the various subunits thereof, and fragments of these subunits. Featured is the novel aspect of discovering the existence of muscle-type receptors in a human neuronal line and the consequent use of such receptors to assay the effects of agents that might affect acetylcholine receptors in human skeletal muscles.

Description

Docket 47929 (Lindstrom) MUSCLE NICOTINIC ACETYLCHOLINE RECEPTORS:
THEIR CHARACTERIZATION, ~REPARATION AND
USE AS DIAGNOSTIC SCREENING REAGENTS

Acknowledgment This invention was made with U.S. Government support in the form of several grants from the National ,! Institutes of Health and the United States Army.

, 10 Related A~lications References made to U.S. Patent Application Serial J. Number 170295, filed 18 March 1988, and to U.S. Serial Number 816383, filed 6 January 1986, published as WO
87/04251 on 16 July 87, the entire disclosures of each of which applications being hereby incorporated by express reference herein.
~7 Field of the Invention The present invention relates generally to the isolation and characterization of certain muscle nicotinic acetylcholine receptors, to their preparation, particularly using recombinant DNA technology, and to their use as diagnostic tools in the screening of materials that may have an effect on receptor function.
More particularly, the present invention i6 directed to a family of mammalian muscle nicotinic acetylcholine receptors, most particularly human muscle nicotinic acetylcholine receptors, and to their various subunit structures, individually or collectively making up the biological function of the parent muscular acetylcholine receptor species.
Novel aspects relating to the preparation of such muscle nicotinic acetylcholine receptor entities, ~ including certain novel DNA isolates encoding same, ¦ expression vectors operably harboring these DNA sequences .~ .

and hosts transfected with said vectors are included within the scope of this invention.
Most particularly, the present invention concerns the use of these muscle nicotinic acetylcholine receptors for screening various materials including organic molecules that may have a negative or positive effect on the function of the muscle nicotinic acetylcholine receptor, so as to devise novel methods of assay analysis or novel drug entities for the treatment of certain diseases or conditions that demonstrate symptoms causal to an impairment of muscle nicotinic acetylcholine receptor function or other materials that do not result in an impairment of muscle nicotinic acetylcholine receptor function. For these purposes, the muscle nicotinic acetylcholine receptors are isolatable from the known TE671 cell line, as expressed therein, or are prepared via harnessed recombinant DNA technology.
The present invention is further directed to the identification, preparation and use of certain monoclonal antibodies in the determination of the structure of the ` muscle nicotinic acetylcholine receptors of this . invention.
This invention, related as it is to muscle nicotinic acetylcholine receptors, is distinct from that for the so-called neuronal nicotinic acetylcholine receptors, as the receptors of the present invention bind alpha bungarotoxin (~-Bgt) and are composed of four subunit types whereas the neuronal acetylcholine receptors do not bind ~-Bgt and are composed of two subunit types.
Although all of these proteins have been described as belonging to the same gene super-family, this characteriza~ion seems to be based on historic, evolutionary comparisons in reference to some subunit cDNA homologies rather than to an overall similarity in structure and especially function. The muscle nicotinic acetylcholine receptors of the present invention have been found to have different immunological, biochemical, ., `:

20034~;9 and pharmacological properties compared with the neuronal acetylcholine receptors. This discovery based on the observation of the existence on muscle-type receptors in a neuronal line is a novel aspect of the present invention as described with more particularity infra.

Background of the Invention The neuron muscular junction has long been a focal point of neuroscience research, primarily because of its accessibility to biochemical and electrophysiological techniques and because of its elegant, relatively well-defined structure and function. Much of this research has concerned acetylcholine receptors because they are a critical link in transmission of signals from nerves to muscles. Action potentials propagated along a motor nerve axon from the spinal cord depolarize the nerve ending causing it to release the transmitter chemical acetylcholine. Acetylcholine binding to receptors in the postsynaptic membrane of the muscle triggers the brief opening of a cation channel through the receptor molecule ` and across the postsynaptic membrane. The resulting flow of cations across the membrane triggers an action potential that is propagated along the surface membrane of the muscle, thereby completing neuromuscular transmission and ultimately causing contraction of the muscle.
Biochemical and molecular genetic studies of acetylcholine receptor structure began with studies of fish electric organs because the organs contained much larger amounts of receptor than was available in muscle.
The structure of muscle type acetylcholine receptors has evolved very conservatively from fish to man, hut the evolution of neuronal acetylcholine receptors apparently diverged early and proceeded rapidly to yield a number of , receptor subtypes. Research on neuronal acetylcholine receptors revealed cDNAs that encode some of the subunits : of neuronal nicotinic receptors are referred to as ~2, ' ~' .
': ' ~ '' ' . ', .' ~00;~4~9 ~3, ~4, ~5, and ~2 and ~3. The results of this research have been accumulated in the patent application above identified as U.S. Serial Number 170295. Reader's access to this related, but distinct, application would reveal that this research also has been collected in certain scientific literature publications that are referenced in said patent application. See also Lindstrom, et al., Neuronal Nicotinic Receptors, Bardard, et al., Eds., NAT0-ASI Series, Springer-Verlag, Heidelberg, "Structure of Neuronal Nicotinic Receptors"; Whiting et al., "Characterization of Bovine and Human Neuronal Nicotinic Acetylcholine Receptors Using Monoclonal Antibodies," J.
Neuroscience 8, 3395 (1988); and Lindstrom, et al., ~ ~ = =. . .
"Molecular Studies of the Neuronal Nicotinic Acetylcholine Receptor Family," Molecular Neurobioloay 1, 281 (1987).
In these documents, the nicotinic acetylcholine receptors from muscles and nerves have been described as components of a gene superfamily. This description was based upon the members having the common property of - having a ligand-gated ion channel formed from multiple homologous subunits. However, although the neuronal acetylcholine receptors described in the aforementioned patent application have been found herein to exhibit sequence homologies with the muscle nicotinic acetylcholine receptors of the present invention, this serves merely as evidence of a common evolutionary - precursor, and only that, because the two classes of acetylcholine receptors have been found by the work underlying the present invention to differ substantially and surprisingly in subunit structure, pharmacological properties, immunological properties, electro-physiological properties, and in respective functional roles. Thus, the muscle nicotinic acetylcholine receptors of the present invention shall enjoy uses separate and distinct from their neuronal acetylcholine receptor counterparts.

';.

X00;~4~9 The present invention is predicated on these separate, distinct properties of the muscle nicotinic acetylcholine receptor class ligand-gated ion channel forming receptors. Indeed, the distinctness of these two classes of receptors is underscored by research evidence demonstrating that antibodies to muscle nicotinic acetylcholine receptors in the serum of patients with myasthenia gravis ~MG) do not bind to acetylcholine receptors from human brain, even though they are homologous. Similarly, there was no common binding of serum from patients with various other syndromes.
Additionally, there was no binding of any serum to the ~-Bgt binding protein from human brain. This data is consistent with other data using antibodies to acetylcholine receptors from both muscle and brain in demonstrating that the acetylcholine receptors in brain is antigenically distinct from that in skeletal muscle.
See Whiting et al., J. Neuroimmunoloay 16, 205 (1987).
Some work has additionally been published on the cloning and sequence analysis of DNA of certain subunits of a muscle acetylcholine receptor gotten from the human genome. See Noda et al., Nature 305, 818 (1983) --~subunit-- and Shibahara et al., Eur. J. Biochem. 146 15 (1985) -- gamma subunit. However, no expression results were documented. Further, sequencing of a subunit provides precious little, if any, information on which of several may be essential for functionality of the muscle nicotinic acetylcholine receptor.
Additionally, nondefinitive chromatographic preparations of nicotinic acetylcholine receptors extracted from human muscle have been reported. See Momoi et al., J. Biol Chem. 257, 12757 (1982). The yields and purities were sufficient for certain biochemical studies but the presence of contaminants could not be excluded.
Finally, Claudio et al., Science ~, 1688 (1987), report on a stable expression of a Torpedo acetylcholine ' .. . . .
- , . . .; ,' .

Z003~59 receptor in a mouse fibroblast cell, opening the possibility of further cell biology studies of this species distinct receptor.
The aforecited patent application Serial Number 816383 served as a starting point for the present invention underlying this application. In said Serial Number 816383 there is described an assay for MG which is an improvement of those described in U.S. Patents 4202875 and RE 30059. The Serial No. 816383 invention involves lo immunoprecipitation of a complex of acetylcholine receptor, toxin and a radioactive isotope label being used as an assay tool to detect and measure the amounts of autoimmune antibodies in MG patients. That invention is based upon the discovery that the acetylcholine receptor derived from a neuronal cell line, namely TE671, can function in the assay surprisingly successfully in comparison with the earlier work which used acetylcholine receptor derived from muscle tissue of human amputees.
See also Lindstrom et al., Myasthenia gravis: "Biology and Treatment," Annals of New York Academy of Sciences 505, 208 (1986).
Thus, that invention demonstrated that the acetylcholine receptors from TE671 cells are immunologically indistinguishable from acetylcholine receptors obtained from human muscle. The cell line TE671 is a human medulloblastoma. See McAllister et al., Int. J. Cancer 20, 206 (1977). It is deposited at the American Type Collection (ATCC) under Deposit No. CRL
8805.
Although that invention demonstrates that an acetylcholine receptor derived from the TE671 cells has proved to be immunologically indistinguishable from human muscle nicotinic acetylcholine, in an assay for MG
detection, the patent application is silent as to whether there are any further distinguishing characteristics between the two molecules. Interestingly, it is silent with respect to comparisons o~ electrophysiological and ' .

- , .

Z00~459 pharmacological properties of the two acetylcholine receptors. Therefore, based upon that invention, it remained unpredictable whether acetylcholine receptor derived from TE671 cells could be used to reliably assay compounds for their affects on the human muscle nicotinic acetylcholine receptor.
This uncertainty and unpredictability is removed by the present invention that demonstrates that muscle nicotinic acetylcholine receptor, whether derived from human muscle tissue or, for example, from TE671 cells, differs electrophysiologically and pharmacologically from neuronal acetylcholine receptors, and because of the discovery of the properties of the muscle nicotinic acetylcholine receptors hereof, they can be used as herein described to assay for derivatives or other materials that may modulate and/or influence acetylcholine receptor activity.
It is an overall object of the present invention to provide science and the medical arts with the structural and electrophysiological and pharmacological properties of a class of mammalian (most notably human) acetylcholine receptors, to describe methods whereby copious amounts of them or their component subunits can be prepared, and to define their use in an assay setting ; whereby various materials can be assayed to determine their effects, if any, on the biological function of these muscle acetylcholine receptor molecule species or their subunits.

Summary of the Invention The present invention is predicated upon the discovery and isolation of sufficient quality and quantity of muscle nicotinic acetylcholine receptor that has enabled the discriminate characterization of human muscle nicotinic acetylcholine receptors, as well as their various subunits, both in terms of physical attributes and their biological function and effect.

' , :`
' .

~003459 These results have enabled in turn the consequence that muscle nicotinic acetylcholine receptors can be employed in a method for screening materials that may modulate muscle acetylcholine receptor activity which comprises challenging said muscle nicotinic acetylcholine receptor species or a functional subunit or associated subunits thereof with one or more of a battery of test materials that can potentially modulate the biofunction of said receptor and monitoring the effect of said material on said receptor in an in vitro setting.
The invention is further directed to an expression vector capable of producing a human muscle nicotinic acetylcholine receptor or a functional subunit thereof, notably the ~ and 6 subunits, which comprises expression control elements operable in the recombinant host selected for the expression of DNA encoding said human muscle nicotinic acetylcholine receptor or functional subunit thereof, and appropriate termination sequences, replication sequences, and other sequences that functionally assist the integration of said expression vector into a recombinant host by transfection, optionally coupled with actual integration into the host's genome.
The invention is further directed to a DNA molecule which is a recombinant DNA molecule or a CDNA molecule consisting of a sequence encoding a certain functional subunit of human muscle nicotinic acetylcholine receptor.
The invention is further directed to substantially ; pure human muscle nicotinic acetylcholine receptor, or a functional subunit thereof selected from the group consisting of an ~, ~, gamma and 6 subunit, obtainable via expression of DNA encoding same in a transfected recombinant host organism, said receptor being thus obtainable in sufficient quantity and quality to enable its use in an in vitro assay suitable for testing the effect of certain extrinsic materials thereon.

.

: : ,., , ,~. . -.-:. : :

Thus, as a primary aspect, the present invention enables assays for extrinsic materials based upon competitive ligand binding to the acetylcholine binding site of the receptor and assays based on alteration of agonist activation of the receptor's cation channel.
Such extrinsic materials may be selected from drug potentials such as muscle relaxants, anesthetics, acetylcholinesterase inhibitors, anthelmintics, etc.;
toxins such as snake venom components, coral, chemical warfare agents, etc.; environmental contaminants such as nicotine, metals, ethanol, etc.; viral components that may use the muscle acetylcholine receptors hereof expediently in the infection process; and other materials that could effect the muscle-neural junctions.
Potential uses predicated on the present invention are in pharmacology including 1) developing improved surgical muscle relaxants; 2) developing improved drugs specific for muscle acetylcholine receptors that do not have an effect on other nicotinic acetylcholine receptors; 3) developing improved drugs for neuronal nicotinic receptors that do not effect human muscle nicotinic receptors; 4) developing improved acetylcholine esterase inhibitors which do not have an effect on nicotinic acetylcholine receptors; 5) developing improved insecticides which do not have an effect on human muscle nicotinic acetylcholine receptors: 6) studying the effects of various chemical agents on muscle nicotinic acetylcholine receptors; 7) studying the effects of various other drugs containing quaternary and tertiary amines for possible affects on muscle nicotinic acetylcholine receptors; and 8) developing better anthelmintics directed at parasitic nicotinic receptors but without effect on human nicotinic receptors for treating various parasites, for example, the parasite Onchocerca volvulus that causes river blindness and is treatable currently with pyrantel which also affects human nicotinic ACh receptor-. me rOregoing would be ~ ~ -,:: .

~.

Z0034~9 made possible from observations of the effects of a given material on receptors from TE671 cells, for example.
This information would allow the development of new drugs, for example, that are more specific, or less, and therefore have fewer untoward side affects.
Finally the present invention provides the multi-disciplinary characterization of human muscle nicotinic acetylcholine receptors:
1. Electrophysiological studies suggesting that muscle nicotinic acetylcholine receptors from TE671 cells are similar to those of muscle at the single channel level;

2. Immunological studies indicating that the muscle nicotinic acetylcholine receptors are distinguishable from neuronal acetylcholine receptors;

3. 8iochemical studies demonstrating that the muscle nicotinic acetylcholine receptors hereof are composed of four types of subunits;

4. Cell biology studies revealing that muscle nicotinic acetylcholine receptor expression is up-regulated by nicotine, human calcitonin gene related peptide(CGRP), or dexamethasone; and reduced by forskolin, which also inhibits cell division and promotes ; the development of extensive neuron-like processes.
As a starting point herein, the TE671 cell line provides all these advantaqes. The acetylcholine receptor subunit cDNAs characterize the protein permitting its expression in other systems such as bacteria or yeast where greater amounts are producible.
It is contemplated that expressed cDNA fragments will be ~ useful to make affinity adsorbents for specific - plasmapheresis or to synthesize conjugates with toxins that could be used to target the toxins to lymphocytes that are autoimmune to acetylcholine receptors.

:, Z00~459 Detailed Description of the ~V~iQn Data presented herein demonstrates that the human neuromedulloblastoma cell line TE671, for example, expresses nicotinic acetylcholine receptors of the type found in skeletal muscle. Thus, drugs, toxins and related agents can be tested on cultures of TE671 cells, for example, to assay their effects on these receptors.
Because the activity of these receptors can be measured electrophysiologically and by agonist-induced ion flux, use of this cell line, or other (e.g., recombinant) line expressing such receptors, permits assays both of agents .
which bind the acetylcholine binding sites on the receptor and of agents which affect receptor function by binding the other parts of the receptor molecule. Given a source, such as TE671 cells, in which receptor activity can easily be measured, particular assay techniques for measuring ligand binding and receptor function will be evident to those experienced in pharmacological studies.
Also described here are cDNAs for subunits of acetylcholine receptors. These identify these receptors as being of the muscle type. Further, they permit expression of functional receptors, individual receptor subunits, or fragments of receptor subunits in other (recombinant) systems. For some purposes, such as certain functional assays or to obtain large amounts of receptor protein for pharmacological or immunological purposes, it may be advantageous to express intact receptors. For other purposes, such as for use as antigens or components of pharmacological agents, it may be advantageous to express individual subunits or fragments of subunits. Methods of expressing these cDNAs - are generally known to those experienced with molecular genetic techniques. For example, see the methods described by Claudio et al., Su~ra.. Fujita et al., Science 231, 1284 (1986) and Barkas et al., Science 235, 77 (1987).

. .
~" .

~no~59 1. Brief description of the drawings Figure 1 demonstrates that acetylcholine (ACh) induces openings of single acetylcholine receptor (AChR~ channels. Openings were recorded at an applied voltage of 100 mV at 0.5 ~M ACh or 70 mV at 10 ~M ACh.
Figure 2 shows an analysis of single AChR
channel currents. 2a) ~Bgt blocks ACh-induced channel openings. Results from several experiments are summed.
ACh was used at 0.5-50 ~M. ~Bgt was used at 0.04-0.15 ~M. Membrane voltage was 70-100 mV. 2b) Linear current/voltage characteristics indicate ohmic channels.
The slope conductance was 45 pS. 2c) Analysis of open channel duration suggests that there are frequent short openings and less frequent longer openings. ACh was used at 10 ~M at a membrane potential of 100 mV. The data (noisy cul~e) were well fitted by a sum of two exponentials (smooth curve) (p>0.05). Total number of events analyzed was 2,035.
Figure 3 displays the subunit composition of AChRs from TE671. 3a) AChRs affinity purified from TE671 and Torpedo electric organ have similar subunits.
AChRs (10 ~g) were resolved into their subunits by i electrophoresis on a 10% acrylamide gel in SDS under reducing conditions and stained with Coomassie blue. 3b) Subunits from TE671 AChRs correspond to those of electric organ AChRs by western blotting. Purified TE671 AChR (50 ng/lane) was resolved into subunits by electrophoresis and then blotted onto paper. Each lane was inc~bated with the indicated antibody, mAb 61 [Tzartos et al., J.
Biol. Chem ~, 8635 (1981)] and mAb 111 [Tzartos et al., J. Neuroimmunol. 10, 235 (1986)] at 10 nM, and gamma and anti-subunit sera [Lindstrom et al., Biochemistry 18, ~ 4470 (1979)] at 1 nM. Bound antibodies were localized by ; autoradiography using 12sI mouse anti-rat IgG. 3c) Affinity labeling with 3H-M8TA identifies the ~ subunits of AChR from TE671 as forming the ACh binding site. 3d) Poly A mRNAs for the four subunits of TE671 AChR are . ~ ., ' -:
, ~

2003~59 detected by high stringency hybridization with cDNAs for mouse muscle AChR ~, ~, gamma, and ~ subunits.
Figure 4 displays a comparison of deduced amino acid sequences for muscle AChR ~ subunits among species. Amino acids conserved in all five species are highlighted. Ml through M4 indicate hydrophobic sequences. The N-glycosylation site on ~ subunits is indicated by ~. Cysteines 192 and 193 are marked by ACh.
Figure 5 shows the nucleotide sequence and deduced amino acid sequence of a TE671 cDNA clone coding ; for the human muscle AChR ~ subunit. The mature protein starts at position +1. The cDNA clone 6.4 extends 124 nucleotides further 5'.
Figure 6 shows a comparison of deduced amino acid sequences for AChR ~ subunits among various species. Numbers indicate amino acid position within Torpedo sequence. Amino acids conserved in all six species are highlighted. Ml through M4 indicate hydrophobic sequences. Potential phosphorylation sites are indicated by Pi. Potential N-glycosylation sites are indicated by ~. The penultimate cysteine, which is thought to be the site of AChR dimerization in Tor~edo, is pointed out by a t.
Figure 7 represents how nicotine, ; dexamethasone, CGRP, and forskolin effect AChR expression in TE671 cells. On day zero lx105 cells were plated in each 3.5 cm dish. On day two media was supplemented as indicated. On day four carbamylcholine-induced ~Rb~
influx was measured on sister triplicate cultures.
Background was determined for each culture conditions and subtracted to give the values shown (average background was 500 cpm). Northern blots using equal amounts of total RNA from other sister cultures were probed successively with 32P-labeled mouse ~, mouse ~, mouse gamma, and human ~ muscle AChR cDNAs. ~2sI~Bgt binding to cell surfaces was measured in a series of cultures.

.. - :

.
" `. ~
.~. ' .

Z003~159 2. General methods and definitions Amino acid identification uses the single-and three-letter alphabets of amino acids, i.e.:
Asp D Aspartic acid Ile I Isoleucine Thr T Threonine Leu L Leucine Ser S Serine Tyr Y Tyrosine Glu E Glutamic acid Phe F Phenylalanine Pro P Proline His H Histidine Gly G Glycine Lys K Lysine Ala A Alanine Arg R Arginine Cys c Cysteine Trp W Tryptophan Val V Valine Gln Q Glutamine Met M Methionine Asn N Asparagine When prepared via recombinant technology herein, the muscle nicotinic acetylcholine receptors hereof are prepared 1) having methionine as the first amino acid (present by virtue of the ATG start signal codon insertion in front of the structural gene) or 2) where the methionine is intra- or extracellularly cleaved, having its ordinarily first amino acid, or 3~
together with either its signal polypeptide or conjugated other than its conventional signal polypeptide, the signal polypeptide or a conjugate being specifically cleavable in an intra- or extracellular environment, or 4) by direct expression in mature form without the necessity of cleaving away any extraneous, superfluous polypeptide, 5) similar extraneous polypeptide located at ` a position 3' of the mature sequence, or 6) as fragments of the AChRs or subunits hereof. In all events, the receptors, in their various forms, are recovered and purified to a level suitable for intended use. See Supra.
As used herein, "AChR" stands for acetylcholine receptor, or more distinctly, muscle nicotinic acetylcholine receptor, and "ACh" for acetylcholine.
As used herein, "biofunctional" means that the thus modified noun refers to an entity that is bioactive, ~0345g i.e, that it functions equivalently as in its biological environment.
Muscle nicotinic acetylcholine receptor is formed from four different subunits (~, ~, gamma and ~).
These are organized in the order ~ , gamma, ~ like barrel staves around a central cation channel. The sites that bind ACh and ~-Bgt and regulate channel opening are located on the ~-subunits, near cysteines ~ 192 and 193.
By the term "human" in reference to the muscle nicotinic acetylcholine receptors hereof is meant the human entities as such as well as derivatives thereof that exhibit the requisite biofunctionality and that differ in one or more amino acids from the human entities. For example, it is contemplated that certain primate (as opposed to lower vertebrate) AChRs would be useful herein particularly as an essential component in the herein described assay system; as such, they are included within the scope of the term.
- Formulations or compositions hereof containing a muscle nicotinic acetylcholine receptor entity as a biofunctional essential component are prepared in accordance with methods known per se in the relevant arts. Thus the formation of pharmaceutical compositions of various sorts are within the ordinary ken of artisans.
Pesticide compositions likewise containing appropriate adjuvants or carriers are likewise well known and documented by standard texts.
"Expression vector" includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operatively linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. "Operative," or grammatical equivalents, means that the respective DNA sequences are operational, that is, work for their intended purposes.

In sum, "expression vector" is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified DNA sequence disposed therein is included in this term as it is applied to the specified sequence. In general, expression vectors of utility in recombinant ~NA techniques are often in the form of viruses or "plasmids" which refer to circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
"Recombinant host cells" refers to cells which have been transfected with vectors constructed using recombinant DNA techniques.
"Extrinsic support medium" includes those known or devised media that can support the cells in a growth phase or maintain them in a viable state such that they can perform their recombinantly harnessed function. See, for example, ATCC Media Handbook, Ed. Cote et al., American Type Culture Collection, Rockville, MD (1984).
A growth supporting medium for mammalian cells, for example, preferably contains a serum supplement such as fetal calf serum or other supplementing component commonly used to facilitate cell growth and division such as hydrolysates of animal meat or milk, tissue or organ extracts, macerated clots or their extracts, and so forth. Other suitable medium components include, for example, transferrin, insulin and various metals.
` The vectors and methods disclosed herein are suitable for use in host cells over a wide range of ; prokaryotic and eukaryotic organisms.
; In addition to the above discussion and the j various references to existing literature teachings, . ~ .

reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques encompassed by the present invention. See, for example, Maniatis, et al, Molecular Cloninq: A Laboratorv Manual, Cold Spring Harbor Laboratory, New York, 1982 and the various references cited therein, and in particular, Colowick et al., Methods in Enzymoloqy yol 152, Academic Press, Inc.
(1987). All of the hereincited publications are by this reference hereby expressly incorporated herein.
The foregoing description and following experimental details set forth the methodology employed initially by the present researchers in identifying and isolating particular muscle nicotinic acetylcholine receptors. The art skilled will recognize that by supplying the present information including the DNA and protein sequences, and characterization and use of these ; receptors, as detailed herein, it is not necessary, or perhaps even scientifically advisable, to repeat these details in their endeavors to reproduce this work.
Instead, they may choose to employ alternative, reliable and known methods. Thus, for example, they may synthesize the underlying DNA sequences for deployment within similar or other suitable, operative expression vectors and culture systems. They may use the sequences herein to create probes, preferably from regions at both the N-terminus and C-terminus, to screen genomic libraries in isolating total encoding DNA for deployment as described above. They may use the sequence information herein in cross-hybridization procedures to ; isolate, characterize and deploy, as above described, DNA
encoding muscle nicotinic acetylcholine receptors of various species, or DNA encoding related (e.g., gene family) receptors or subunits thereof of the same or other species, or to devise DNA for such characterization, use and deployment encoding functionally equivalent receptors or subunits thereof of .

Zt)(~345~

all of the above differing in one or more amino acids from parental (wild-type) species or in glycosylation patterns or in bounded conformational structure.
Thus, in addition to supplying details actually employed, the present disclosure serves to enable reproduction of the specific receptors disclosed and others, and subunits thereof, using means within the skill of the art having benefit of the present disclosure. All of such means are included within the enablement and scope of the present invention.

3. Examples The following examples detail materials - and methods employed in the experimental procedures that follow:
TE671 cells Cultures were grown at 37-C in 90% air 10%
CO2 in Iscove's modified Dulbecco's medium from Irvine Laboratories supplemented with either 10% fetal bovine serum or 5% bovine calf serum. For electrophysiological studies, 104 cells were plated per well in a 24-well plate on 12 mm diameter cover glass slips in medium with 10~
serum. One day later, serum was reduced to 0.01%, and 2 mM L glutamine, 19 ~g/ml insulin and transferrin were added. Electrophysiological studies were done in 115 mM
NaCl, 5 mM CsCl, 1 mM MgCl2, 25 mM glucose, 25 mM HEPES, pH 7.4, 10 mM TEA, and 0.1 mM anthracene-9-carboxylic acid.
'~.
Electrical Recordings Single channel current electrical recordings were performed as described by Sakmann et al., Sinale Channel Recordina, Plenum Press, NY (1983). Recordings were obtained in both the cell attached and the excised patch configurations. Ihe pipettes were fabricated from XOVAR glass (Corning 7052, ID=l.l mm, OD=l.S mm, 70 mm lng) using a vertical pipette puller (David Kopf 700 C, Tu~unga, California). The pipettes were coated with ., :, '''~

'~ , -.

Sylgard-180 tDow Corning) within 40 ~m from the tip and fire polished immediately before use under 320X
magnification. The tip size was adjusted to yield 5-15 Mohms of open pipette resistance when filled and immersed in the buffer described before. The patch pipettes contained the indicated concentration of ACh diluted in the same solution. The cells were observed with an inverted microscope (Nikon-diaphot) using a 40X objective (LWD DL 40XC, Nikon) equipped with Hoffman modulation contrast optics (Modulation Optics, Greenvale, New York).
The microscope was mounted on a vibration isolation table (Micro g Technical Mfrg. Corp., Waltham, Mass.).
A commercially available extracellular patch clamp system was used (LM EPC-5, List Electronics, Darmstadt, West Germany and Medical Systems Corporation, New York). The headstage of the amplifier was mounted on a hydraulic micromanipulator (M0-103N Narishige, Japan).
The signal output from the clamp was recorded on FM tape (Racal 4DS, Hythe, Southhampton, England, bandwidth DC to 5 kHz). All the records were filtered at 2 kHz on an 8 pole Bassel low pass filter (Frequency Devices, 9028LPF, Haverhill, Mass.). The data were digitized at the sampling frequency of 10 kHz in an Indec-L-11/73-70 microcomputer system (Indec, Sunnyvale, California).
conductance levels were discriminated as described by Labarca et al., J. Gen. Physiol 83, 473 (1984).
Histograms of dwell times in the open state and closed states of the AChR channel were analyzed as described in detail previously [Labarca et al., Su~ra and Bioloay J.
47, 469 (1985): Montal et al., Ion Channe~
Reconstitution, C. Miller, Ed., p. 157, Plenum Press, NY
(1986)]. The results of at least five different experiments in each condition are presented infra. All experiments were done at room temperature (22 C).

Preparation of Solubilized TE671 Membrane Extracts TE671 cell cultures were grown in T-flasks for six days and then expanded to two liter (850 cm2) roller bottles in 5~ BCS in Iscove's modified DMEM medium (Irvine Laboratories) with 2.5 ~M dexamethasone. After ten days in culture the cells were harvested after aspiration of media by first rinsing with cold phosphate buffered saline (PBS), pH 7.5, containing 10 mM
iodoacetamide (IAA), 10 mN aminobenzamodine, 1 mM
phenylmethylsulfonaylfluoride (PMSF) to remove the excess media, and secondly by shaking in 25 ml per bottle of 50 mM Tris, 150 mM NaCl, 100 mM KF, 5 mM EDTA, 5 mM EGTA, 5 mM IAA, 5 mM aminobenzamidine, 0.5 mM PMSF, bestatin (10 ~g/ml), Trasylol (10 ~g/ml), soybean trypsin inhibitor (10 ~g/ml), pH 7.5 tbuffer A). The bottles were then rinsed with four volumes of buffer A to remove any remaining cells. The cells were then pelleted by centrifugation at 3000g for 30 minutes. The resulting cell pellet was resuspended in 400 ml of buffer A, lysed by homogenization using a Polytron for 30 seconds, and centrifuged for 30 minutes at lO,OOOg. The membrane pellet was resuspended in 250 ml of buffer A, homogenized, and centrifuged as described in the previous step. The resulting pellet was then extracted for 30 minutes in four volumes of buffer A with 1% Thesit detergent (Boehringer) and 0.05% sodiumdodecylsulfate (SDS), pH 7.5, centrifuged at 140,000g for 30 minutes, and the clarified supernatant was retained.

Purification of the TE671 AChR
~ Bgt was first coupled to Sepharose CL4B at 5.0 mg of protein/ml of gel by a procedure of Kohn et al., Biochem Biophys Res. Comm. 107, 878 (1982). The clarified, solubilized TE671 membrane extract (75-100 ml) ; from, typically, 12 roller bottles, was applied to a 20 ml column of Sepharose CL4B to adsorb any proteins which may nonspecifically absorb to the column, The eluate was ' '' " ;. ' ' ' - , ~

., ,: .

then applied to a 1 ml column of ~Bgt-affinity gel and both columns were washed with 200 ml of the extraction buffer. The affinity column was consecutively washed with 200 ml of buffer A containing 1.0 M NaCl, 0.5%
Thesit, 0.0S% SDS pH 7.5, followed by 150 ml of 10 mM
Tris, 0.1% Thesit, 1 mM NaN3, 10 mM KF, 1 mM IAA, 1 mM
aminobenzamidine, 1 mM EDTA, and 1 mM EGTA pH 7.5 (buffer B). The affinity column was then coupled to a hydroxylapatite (HPT) column (1 ml) and the TE671 AChR
eluted onto the HPT column by recirculating through both columns for 12 hours, 10 ml of buffer B containing 200 mM
carbamylcholine, using a peristaltic pump. After displacement of the bound protein, the HPT column was washed with 200 ml of buffer B and then eluted with 150 mM sodium phosphate, 0.5% Thesit, 1 mM NaN3, 1 mM PMSF, 1 mM EDTA, 1 mM EGTA, 1 mM aminobenzamidine, 1 mM IAA at pH
7.5.

Affinity Labeling TE671 AChR was immobilized on ~Bgt-sepharose and then affinity labeled with 3H-MBTA as previously described by Whiting et al., FEBS Letters 213, 55 ~1987).
Sucrose Gradient Centrifugation Electrophoresis ` Electrophoresis was conducted on acrylamide ; slab gels in SDS using a Laemmli discontinuous buffer system [Laemmli, Nature 227, 680 (1970)]. Polyacrylamide gels were silver stained for protein according to the method of Oakley et al., Analyt. Biochem. 105, 361 (1980). Polyacrylamide gels of radio-labeled protein were autoradiographed for 4-24 hours at -70C using preflashed Kodak X-Omat-AR film and an intensifying screen. Autoradiograms were standardized by using Sigma prestained low molecular weight standards resolved on the same gel. Electrophoretic transfer of proteins from gels to diazophenylthioether (DPT) paper and subsequent :, . , 20034~;9 probing with antibodies were as described by Gullick et al., J. Cell Biochem. 19, 223 (1982)~ After being probed, bound antibodies were detected by incubation with 0.5 nM 12sI-labeled mouse anti-rat IgG (1-3 x 1018cpm/mol) and autoradiography.

Cloning and Sequencing of TE671 AChR ~ Subunit cDNA
A cDNA library was prepared as described by Schoepfer et al., FEBS Letters 226, 235 (1988). The filters were screened under high stringency with the ~450 bp Eco RI-Ava I fragment of cDNA clone BMD451 (Heinemann et al., Nicotinic Acetylcholine Receptor Structure and Function, ~aeliche, Ed., p. 360, Springer-Verlag, Heidelberg) coding for the 114 N terminal amino acids of the mouse AChR ~ subunit. A single positive clone was identified. Plasmid DNA was characterized by restriction enzyme digestion, followed by agarose gel electrophoresis and Southern blot analysis. From the ~3 kb insert, the 5' ~1860 bp Eco-Ava fragment was subcloned into a plasmid vector. Nested deletions were produced by the Exo III/Mung Bean protocol provided by Stratagene.
DNA sequencing was performed using a modification of the dideoxynucleotide chain termination method of Sanger et al., PNAS 74, 5463 (1977).

Regulation of TE671 Expression Cultured cells grown in T-flasks were harvested and 1 x 105 cells were plated in 6-well tissue culture dishes in Iscove's medium containing 10% FCS. After two days, the media was removed and replaced with this medium containing the indicated concentrations of forskolin, nicotine, human CGRP (commercially available) or dexamethasone. Forskolin and dexamethasone were dissolved in 95% ethanol while CGRP was dissolved in PBS.
~ Ethanol or PBS alone had no effect on cell growth or AChR
-~ expression. The cells were grown for two days and the number of ~Bgt binding sites, AChR function, and RNA

..

~o()~ 9 encoding the ~, ~, gamma, and ~ subunits of the TE671 AChR were determined.
The number of ~Bgt binding sites was determined as follows: After two days the medium was removed and the cells were washed three times with 2 ml of Iscove's media. The cells were then labeled for one hour with 0.5 ml of 20 nM 125I~Bgt in Iscove's medium at 37C.
Nonspecific binding was determined by performing the experiments as described, in the presence of 1 mM
carbamylcholine. After one hour, the cells were again washed three times with 2 ml Iscove's medium. The cells were solubilized with 1.5 ml of 0.5 N NaOH, removed, and bound 12sI~Bgt determined by gamma counting.
AChR function was measured by carbamylcholine-induced influx of ~Rb~ using a procedure of Robinson et al., Molec. Pharm. 27, 409 (1985). Briefly, after two days of growth in the presence or absence of the various indicated effectors, the media was removed and the cells washed three times with 2.0 ml Iscove's. After the third wash, the cells were incubated for one hour in 0.5 ml Iscove's to allow recovery from desensitization of AChRs by the effectors. Media was removed and the cells washed two times with 2.0 ml 0.5 M sucrose, 5 mM KCl, 10 mM
glucose, 1.8 mM CaCl2, and 15 mM HEPES pH 7.4. The cells were then washed with 0.5 ml of the same buffer with 2 mM
ouabain for 20 seconds to inhibit Na~-K~ ATPases. The buffer was removed and ~Rb' uptake was initiated by exposing cells to 0.5 ml of the ouabain buffer containing 5 ~Ci/ml of ~Rb~ with 1 mM carbamylcholine. Control experiments were performed as described, in the absence of carbamylcholine. Uptake was terminated after 30 seconds by aspirating the radioactive solution and rapidly washing three times with 3 ml of 0.3 M NaCl, 5 mM
KCl, 1.8 nM CaCl2, 10 mM glucose, and 15 mM HEPES, pH 7.5.
The washed cells were solubilized with 1.5 ml 0.5 N NaOH
to permit ~Rb~ uptake and protein determination.
Rad1oactivity was determined by liquld ecintillation ;~003~S9 counting of the solubilized cells. Results were normalized as described for the determination of ~Bgt binding sites.
Total RNA was isolated by the guanidine thiocyanate-CsC1 procedure of Chirgwin et al., Biochemistry 18, 5294 (1979). The amount of RNA isolated was quantitated by A260, and equal amounts of RNA from each treatment were size-fractionated by agarose gel electrophoresis containing formaldehyde. The gel was transferred to nylon membranes and probed (Figure 7) with cloned cDNA inserts for the ~, ~, and gamma subunits of the mouse muscle AChR [Heinemann et al., Nicotinic Acetylcholine Receptors Structure and Function, A.
Maelicke, Ed., p. 360, Springer-Verlag, Heidelberg (1986)], and the ~ subunit probe was derived from the cDNA clone for TE671 ~. Hybridization was conducted under highly stringent conditions: 42C, 50% formamide, 5 x SSPE, final washing at 65C, 0.3 x SSPE (where 5 x SSPE is 0.9 M NaC1, 0.5 mM Na phosphate pH 7.4, 5 mM
EDTA). Autoradiography was performed as described above.
Poly A' RNA was prepared from total RNA by oligo-dT column chromatography. The mRNA species for ~, ~, gamma, and ~ was identified as above using muse muscle cDNA probes (Heinemann et al., Supra, 1986).
Experimental detail and discussion using the procedures outlined above follow:
AChRs solubilized from TE671 cells and labeled with l2sI~Bgt are not immune precipitated by a 400-fold molar excess of antiserum to the ~Bgt-binding protein purified from chicken brain. This high titer antiserum precipitates 5 ~moles of ~Bgt binding sites from chicken brain per liter of serum and crossreacts 0.8% with the - ~Bgt-binding protein from human brain. The antiserum also shows no reaction on western blots of purified TE671 ` AChR under conditions where antisera to AChR purified from TE671 label corresponding subunits from AChRs of TE671 and Torpedo electric organ. This data, along with .

- ~ ' :

. .

2003~59 the fact that mAbs like 35, 2U3 and 210, and MG patient autoantibodies bind AChR from TE671 but not ~Bgt-binding proteins from human brain, suggest that TE671 AChRs are not identical to the common ~8gt-binding proteins from human brain.
AChRs solubilized from TE671 cells and labeled with 12sI~Bgt were also not immune precipitated by a 100-fold molar excess of mAbs 290, 293, or 299 which react with AChRs from human brain that have high affinity for nicotine but do not bind ~gt. Thus, TE671 AChRs are also different from AChRs detected in adult human brain.
These AChRs can be blocked by ~Bgt by measuring carbamylcholine-induced ~Rb' influx. The patch clamp technique was used to study AChR activity electrophysiologically at the single channel level. To record only ACh-activated channels, several other channel types present in these cells are blocked pharmacologically: K~ channels are eliminated by adding tetraethylammoniumchloride (TEA) and removing X' from the medium; Ca'~ channels are eliminated by removing Ca~ from the medium; and Cl channels are blocked with 0.1 mM
anthracene-s-carboxylic acid.
ACh induces bursts of AChR channel openings (Figures 1 and 2). At 0.5 ~M ACh the channels are open 3.3% of the time, whereas at 10 ~M ACh this increases to 8.8% (Figure 1). opening of TE671 AChR channels induced by ACh is blocked by ~Bgt (Figure 2a). TE671 AChR
channels exhibit a linear current/voltage relationship in the range 10-100 mV (Figure 2b) with a single-channel conductance (gamma) of 44-45 pS (Figure 1). The majority of channel openings are brief (65% have a time constant [theta] of 0.82 ms), whereas a minority of the openings are more prolonged for 35% theta=3.3 ms) (Figure 2c).
The duration of opening and magnitude of conductance are not affected by the concentration of ACh in the range 0.5-20 ~M or by voltage in the range 50-100 mV. Blockage of function by ~Bgt is the critical characteristic ..
:

distinguishing ACh~s on TE671 from those on neurons, since other electrophysiological properties are similar for neuronal AChRs which do not bind ~Bgt.
AChRs affinity purified from TE671 cells on an ~Bgt affinity column consist of four polypeptides corresponding to ~, ~, gamma, and ~ subunits of AChR from Torpedo electric organ by apparent molecular weights (42,000; 52,500; 55,000; and 62,000) (Figure 3a) and also by antibody labeling on western blots (Figure 3b). The ACh binding site is formed by ~ subunits, as shown by affinity labeling with 4-(N-maleimido)benzyltri-methylammoniumiodide (MBTA) (Figure 3c), an antagonist which specifically blocks ACh binding and labels cysteines 192,193 on the ~ subunit. A typical purification is reported in Table 2. The specific activity of unpurified AChR from TE671 cells (0.09 nmol/g tissue) is 5% that of Torpedo electric organ and 27 times that of fetal calf muscle. Immunoaffinity chromatography on mAb 210 yields a preparation of similar purity which ~ 20 is unable to bind ~Bgt efficiently due to the use of a ; denaturing rather than a competitive elution step.
TE671 cells contain poly A~ mRNAs corresponding to the four kinds of subunits of muscle AChR which are detectable under high stringency hybridization conditions with probes for the ~, ~, gamma, and ~ subunits of AChR
from mouse muscle (Figure 3d). ~ Subunit mRNA is present in 3-5 fold higher concentration than the other subunit mRNAs.
The sequence of a cDNA for the ~ subunits of AChR from TE671 (Figure 4) exhibits 97% sequence identity with ~ subunits from bovine muscle, 95% sequence identity with ~ subunits from mouse, 82% identity with ~ subunit for Xenopus and 81% identity with ~ subunits from Torpedo. In particular, ~ subunits from TE671 resemble those from other species in exhibiting four hydrophobic sequences, an N-glycosylation site, and cysteines at positions 192, 193 which, in the case of Torpedo, have , been shown to react with affinity labeling reagents for the ACh binding site.
The sequence of a cDNA for the ~ subunits of AChR from TE671 is that also expected for ~ subunits of muscle AChRs (Figures 5 and 6). Comparison of the amino acid sequence of TE671 AChR ~ with that of ~ subunits form other species reveals 91~ identity to calf, 90%
identity to mouse, 72% identity to chicken, 68% identity to Xenopus, and 61% to Torpedo. It exhibits 30% sequence identity in the mature protein to ~ subunits from TE671, showing that the subunits forming the AChR are homologous. In comparison with ~ subunits of other species, the ~ subunits from TE671 show conservation of four hydrophobic sequences, three putative N-glycosylation sites, and three putative phosphorylation sites. Like ~ subunits from muscle of other species, ~
subunits from TE671 lack a cysteine penultimate to the C-terminus which is found in ~ subunits of AChRs from Torpedo electric organ.
Treatment of TE671 cells with nicotine, dexamethasone, or hCGRP results in an increase in the observed ~Bgt binding to AChRs on the cell's surface (Figure 7). Nicotine and dexamethasone result in an elevation in the number of functional AChRs, while only dexamethasone causes an increase in the amounts of most AChR subunit RNAs. Although hCGRP upregulates ~Bgt binding, AChR function is reduced. The induction of AChRs in TE671 cells appears to occur by more than one mechanism. Both transcriptional regulation of synthesis (in the cases of nicotine and hCGRP) seem to be involved.
The means by which AChR synthesis is regulated in TE671 cells and in muscle cells may differ because, whereas nicotine upregulates the amount of AChR in TE671 cells, agonists downregulate the number of AChR in muscle cells.
Treatment of TE671 cells with forskolin causes a decrease in the number of functional AChRs to :

~;' ' ' " , ' ~
. . . .
.~ , .

~0~ 459 background levels, which is accounted for by the reduction of ~Bgt binding and AChR subunit mRNAs (Figure 7). Forskolin also appears to inhibit cell division and result in the formation of extensive neuronal-like projections. This effect, however, is reversible.
These results suggest that if the effects of forskolin are mediated by an elevation in cAMP concentration, then the increase in AChR induced by hCGRP in TE671 must not be mediated by increased cAMP and may involve some other second messenger. cAMP-induced phosphorylation and desensitization of AChRs could explain the lower number of active AChRs on the surface of hCGRP-treated TE671 cells. Forskolin treatment of TE671 cells does not induce synthesis of neuronal nicotinic AChRs, since treated cells do not bind [3H]nicotine with high affinity or mAbs specific for human brain AChRs. Hybridization signals are not detected when northern blots of TE671 RNA
are hybridized at high stringency with the cDNA probe, ~4 which codes for the ACh-binding subunit of the AChR from rat brain which has high affinity for nicotine.

., . :
.. ~ :

: -:
:

'~(3~)3~59 Table 1. mAb Crossreactivity Between Human Muscle and TE671 Receptor.

Titer (~M) Specificity Immunogen mAb Human Muscle TE671 ________________________________________________________ MIR Human muscle 192 12.0 22.5 AChR
MIR Human muscle 196 0.24 0.74 AChR
BC3Hl and MIR, ~ fetal bovine 210 7.1 7.0 muscle MIR, ~ Human muscle 203 1.4 2.2 - AChR
MIR, ~ Human muscle 207 7.3 5.1 . AChR
~ Torpedo ~ antisera 0.005 0.008 ~ Torpedo ~ 111 0.70 1.2 ~ Torpedo ~ antisera 0.004 0.005 gamma Bovine muscle 66 0.89 0.72 AChR
gamma Torpedo gamma antisera 0.0015 0.001 Torpedo ~ 137 0.52 0.74 Toroedo ~ antinera 0.002 0.001 .

;~003~59 Table 2. Purification of AChR from 20 g (12 Roller Bottles) of TE671 Cells.
________________________________________________________ AChR ~Bgt lZsI~Bgt Volume Protein Binding Sites Specific Fraction (ml) (mg) Mpmol) % Activity (pmol/mg protein) ________________________________________________________ Initial extract: 100 570 1804 100 3.16 Unbound to ~Bgt affinity : column: 100 380 702 39 Wash steps:650 180 342 19 Affinity column eluate: 6 0.100 776 43 7,800 ~.

.~ .

. ~:
~, ~,.
.
:`

:~........................... ' .: ' , ' ' . ; '' ,, ' -:. . .
~' ' , .
. .

The foregoing description details specific methods that can be employed to practice the present invention. Having detailed specific methods initially used to identify, isolate and use muscle nicotinic acetylcholine receptors hereof, and the further disclosure as to protein and DNA sequences, the art sXilled will well enough know how to devise alternative, reliable methods for arriving at the same information and for extending this information to other species and other related receptors. Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope hereof; rather, the ambit of the present invention is to be governed only by the - lawful construction of the appended claims.

.- .

~ ~ .
`. :` ' :
.

.

:

Claims (34)

1. An assay for screening and identifying materials having a modulating effect on human muscle nicotinic acetylcholine receptor function comprising the steps comprising:
a.) providing a human muscle nicotinic acetylcholine receptor species or functional subunit or associated subunits thereof, in a form having quality and quantity sufficient to enable its use in measuring extrinsically induced modulated biofunction thereof, b.) challenging said form of human muscle nicotinic acetylcholine receptor species or functional subunit or associated subunits thereof with one or more of a battery of test materials that can potentially modulate the biofunction of said muscle nicotinic acetylcholine receptor species or functional subunit or associated subunits thereof, c.) monitoring the effect of said test material on the biofunction of said human muscle nicotinic acetylcholine receptor species or functional subunit or associated subunits thereof, and d.) selecting candidates from the battery of test materials capable of modulating the biofunction of said human muscle nicotinic acetylcholine receptor species or functional subunit or associated subunits thereof.

2. An assay according to Claim 1 further comprising the additional step or steps following step d.), comprising:
e.) employing said candidate in the preparation of a composition containing said candidate as an essential component, said composition being useful to impart its modulating biofunction properties on a human muscle acetylcholine receptor when it is contacted in vivo with said receptor.

3. An assay according to Claim 2 further comprising the additional step or steps following step e.) comprising:
f.) contacting said composition with a human subject.

4. The assay according to Claim 3 wherein said step f.) contacting is accomplished via administration to said human subject.

5. The assay according to Claim 4 wherein the administration is parenteral.

6. The assay according to Claim 4 wherein the administration is by ingestion.

7. The assay according to Claim 4 wherein the administration is by inhalation.

8. The assay according to any one of Claims 2 to 7 wherein said composition is a pharmaceutic.

9. The assay according to Claim 8 wherein said pharmaceutic is a muscle relaxant.

10. The assay according to Claim 8 wherein said pharmaceutic is an anesthetic.

11. The assay according to Claim 1 wherein said candidate is a toxic chemical agent.

12. The assay according to Claim 1 wherein said candidate is a pesticide.

13. The assay according to Claim 12 wherein said pesticide is an herbicide.

14. The assay according to Claim 12 wherein said pesticide is an insecticide.

15. An assay according to Claim 1 wherein said muscle nicotinic acetylcholine receptor is provided according to step a.) from TE671 cell source.

16. An assay according to Claim 1 wherein said human muscle nicotinic acetylcholine receptor is provided according to step a.) from recombinant cell source.

17. In an assay according to any one of the preceding claims wherein said human muscle nicotinic acetylcholine receptor has a TE671 cell source.

18. In an assay according to any one of the preceding claims wherein said human muscle nicotinic acetylcholine receptor has a recombinant cell source.

19. In an assay according to Claim 17 wherein said muscle nicotinic acetylcholine receptor is the delta subunit of human muscle nicotinic acetylcholine receptor.

20. A DNA molecule that is a recombinant DNA
molecule or a cDNA molecule consisting of sequence encoding the .alpha., .beta., gamma and .delta. subunits of muscle nicotinic acetylcholine receptor.

21. A DNA molecule that is a recombinant DNA
molecule or a cDNA molecule encoding the .delta. subunit of muscle nicotinic acetylcholine receptor.

22. An expression vector operatively harboring DNA encoding muscle nicotinic acetylcholine receptor.

23. A recombinant host cell transfected with an expression vector according to Claim 22.

24. A recombinant host cell according to Claim 23 which is an E. coli strain.

25. A recombinant host cell according to Claim 23 which is an eukaryotic line.

26. A cell culture comprising cells according to Claim 23 and an extrinsic support medium assuring the viability of said cells.

27. A process which comprises the preparation of a muscle nicotinic acetylcholine receptor polypeptide which comprises expressing in a recombinant host cell transfecting DNA encoding said polypeptide.

28. A process which comprises recovering and purifying muscle nicotinic acetylcholine receptor or functional subunit or associated subunits thereof to a form having quality and quantity sufficient to enable its use in measuring extrinsically induced modulated biofunction thereof.

29. The process according to Claim 28 performed from a TE671 cell source.

30. The process according to Claim 28 performed from a recombinant cell source.

31. Muscle nicotinic acetylcholine receptor or functional subunit or associated subunits thereof in a form having quality and quantity sufficient to enable its use in measuring extrinsically induced modulated biofunction thereof.

32. Muscle nicotinic acetylcholine receptor according to Claim 31 which is human muscle nicotinic acetylcholine receptor.

33. Muscle nicotinic acetylcholine receptor according to Claim 32 which is the .delta. subunit.

34. Muscle nicotinic acetylcholine receptor according to Claim 32, as obtained by expression from a recombinant host cell, in biofunctional form comprising the subunits assembled in .alpha., .beta., .alpha., gamma, .delta. order.