CN107596341A - L-type valtage-gated calcium channel specific polypeptide excitomotor and inhibitor - Google Patents
L-type valtage-gated calcium channel specific polypeptide excitomotor and inhibitor Download PDFInfo
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Abstract
The present invention proposes purposes of the polypeptide in medicine is prepared and the method for screening medicine.The medicine is used to regulate and control L-type valtage-gated calcium channel, and the polypeptide has SEQ ID NO：Amino acid sequence shown in 1~8 any one.The present invention polypeptide can specific regulatory control LTCC passages, so as to influence neuronal excitation transcription couple, further function as regulation and control neuronal development effect.
Description
Technical field
The present invention relates to biomedicine field.In particular it relates to L-type valtage-gated calcium channel specific polypeptide class
Activator and inhibitor.
Background technology
L-type valtage-gated calcium channel (L-type calcium channel, LTCC, also referred to as CaV1 passage) it is a kind of wide
Calcium channel of the general expression in the important hat energy organ such as nervous system, heart.The converter important as internal Electrical excitability, LTCC
The electric signal as caused by action potential on cell membrane can be converted into intracellular Ca2+ oscillations.Calcium ion passes through CaV1 passage enters
Enter in cell, as the second messenger of electric signal, many different cellular activities can be originated.Particularly in neuron,
CaVExcitements such as 1 passage and cynapse transmission, gene regulation-transcription couples process are closely related.It is god that excitement-transcription, which couples process,
An important biochemical process through meta function and growth adjustment, at present Main Viewpoints think, by CaVThe Ca2+ oscillations that 1 passage flows into
Can high special effectively cause excitement-transcription to couple process, detailed process is as follows：Neuron is after by electro photoluminescence or chemical stimulation
Cause film potential to rise (neuronal excitation), and then open CaV1 passage, Ca2+ oscillations pass through CaV1 passage is flowed into neuron, is swashed
Living and CaVThe calmodulin (Calmodulin, abbreviation CaM) that 1 passage combines in advance, and the specific activation near passway
CaMKII, the CaMKII after activation, which enter in nucleus, causes gene transcription factor CREB phosphorylation (to be referred to as CaMKII-
PCREB signal paths), activating ELK 1 B and the regulation and control for influenceing gene transcription level, and then influence neuronal development and function can
Plasticity.This process is the significant process of neuron autogenous control, if the process occurs lesion and can cause many mental illnesses.
At present, CaV1 passage has found the genetic association that height be present with five kinds of main spirits diseases, including self-closing disease, note
Defect of anticipating more dynamic obstacle, the two-way disturbance of emotion, major depressive disorder and schizophrenia.Ca simultaneouslyV1 passage have also discovered a variety of points
Mutation causes mental illness, such as timothy syndrome etc..LTCC is also caused to turn into important with the highlights correlations of mental illness
Drug target.Main Viewpoints are thought by adjusting the feasible way that LTCC Ca2+ oscillations are improvement mental disorders, existing
The interference method of LTCC Ca2+ oscillations concentrates on the activity of suppression/enhancing calcium channel, and then regulates and controls Ca2+ influx, for LTCC designs
Specific inhibitor and activator turn into potential antipsychotics.The existing history for many years of the exploitation of this kind of medicine, all multiple medicines
Thing has become Clinical practice common drug, such as the inhibitor such as Nimodipine nimodipine, isradipine isradipine
It is widely used in the activators such as cardiovascular and cerebrovascular disease etc., BayK8644 to be applied to strengthen myocardial contraction etc..But due to nervous system
Complexity, influence of these medicines to nervous system, the influence particularly coupled to excitement-transcription, certain dispute also be present.
Such as it has been reported that although activator BayK8644 can strengthen CaVSize of current after 1 opening, but do not detected in neuron
The growth course of CaMKII mediations；And in addition it has been reported that inhibitor isradipine can help to mitigate deterioration of neurons mistake
Journey.Utilize existing CaVRegulation excitement-transcription that 1 channel agonist and inhibitor there is no method controllable couples process.So far also still
There is not the report for being directed to the specific regulatory control medicine that excitement-transcription couples.
The content of the invention
It is contemplated that at least solves at least one technical problem present in prior art.
The present invention is the following discovery based on inventor and completed：
Existing scheme is based primarily upon regulates and controls passage Ca2+ influx by LTCC specific agonist and activator, and then it is expected
Regulation and control excitement-transcription couples.But in practical application, these medicines show complicated pharmacological action, and (especially medicine is made for a long time
Under), may not necessarily Effective Regulation excitement-transcription couple.
LTCC activators on sale and inhibitor are generally screened by large-scale medicine and obtained on the market at present, then verify it
Drug target specificity.Typically, the acquisition of inhibitor is easier to realize, small molecule class inhibitor typically directly acts on logical
Near road junction.By contrast, the operation principle of LTCC activators is not entirely understood, its quantity well below inhibitor quantity,
Widely used activator includes BayK8644 and two kinds of FPL-64176, and acquisition has great contingency, such as BayK8644
It is exactly an excitomotor serendipitous when inhibitor is screened.
As small molecule class medicine, inventor is with most representative two kinds of medicines：Inhibitor isradipine (Chinese names
Isradipine) and activator BayK8644 exemplified by, dependence test (Fig. 1, Fig. 2) has been done to medicine effect and neurotoxicity respectively.
Generally, it is considered that the activity of enhancing neuron L-type calcium channel, such as the means such as the stimulation of high potassium, electro photoluminescence are favourable
Coupled in starting excitement-transcription, and then promote neuronal development.But at some in particular cases, such as in the nervous system of lesion
In many sacred diseases such as alzheimer syndrome, parkinson's syndrome, increase activity causes deterioration of neurons on the contrary
Aggravation, be advantageous to therapeutic effect on the contrary after adding inhibitor.To understand fully this micromolecular excitomotor and inhibitor to normality god
Influence through member, the cortical neuron that inventor has separated newborn mice are gone forward side by side the cultured in vitro of 1 week or so behavior phase, cultivated
The 5th day transfection YFP be subsequently observed with facilitating for neuron morphology, the 7th day of culture is observed using immunofluorescence means
Excitement-transcription couple in significant signal path --- pCREB signals, or directly observe neuronal development form.At the 5th day
And 1 μM of BayK8644 and 5 μM of isradipine is separately added between the 7th day and is stimulated 1 hour, 1 day or 2 days and (Fig. 1 .a, is schemed
2.a), to observe under medicine different stimulated duration, the influence to pCREB signals and downstream developmental morphology.Experiment is found, with compareing
Group is compared, and activator BayK8644 has to neuron pCREB signals under the stimulation of journey in short-term (1 hour) and is obviously improved effect
Fruit, but under the stimulation of long time-histories (1 day or 2 days), BayK8644 loses the effect (Fig. 1 .b) to pCREB signal boosts；From nerve
From the point of view of first developmental morphology, journey BayK8644, which is stimulated, in short-term shows the increase effect slight to neurite total length, but long
Time-histories BayK8644 stimulations cause cortical neuron development and are damaged (Fig. 1 .c) on the contrary, show significant neurotoxicity.Suppress
Agent isradipine has the inhibition of conspicuousness, but long time-histories (1 to pCREB signals in the case where journey in short-term stimulates for (1 hour)
It or 2 days) stimulate under, neuron pCREB signals show the process gradually recovered, and under the stimulation duration of 2 days, neuron is
It is recovered to arrive the level (Fig. 2 .c) suitable with control group；From the point of view of neuronal development form, the stimulation of journey to long time-histories in short-term is also
The process gradually recovered equally is showed, but does not cause conspicuousness to destroy (Fig. 2 .b) under overall process to neuronal development.
In summary, current existing CaV1 activator and inhibitor is coupled to neuronal excitation-transcription and development impact
The pharmacological action of complexity is shown, its regulation and control result is often inconsistent with being generally understood that, and importantly, there is no one kind to be directed to
LTCC medicine is effectively facilitated neuronal excitation-transcription and coupled and growth course when can be long.
In view of this, to avoid the uncertainty that large-scale medicine screening zone is come, it is contemplated that being different from from another kind
The mechanism of action of existing activator (such as BayK8644) and inhibitor (such as isradipine) sets out, and is designed to specific regulatory control
LTCC passages, and efficiently and controllably regulation and control neuronal excitation-transcription is coupled and developed the LTCC passages after regulating and controlling when can be long.
Inventor designs specific regulatory control polypeptide by further investigation from the molecular mechanism of LTCC Ion channel kinetics regulation and control.LTCC
In the presence of a kind of special dynamics regulation process, referred to as calcium relies on inactivation regulation and control (Calcium dependent
Inactivation, abridge CDI), what it regulated and controled that result can influence LTCC enters calcium ability.There is article to show at present, calcium, which relies on, to be lost
Living is in the nature to be strengthened by the channel function of calmodulin (Calmodulin, abridge CaM) mediation, and calmodulin is that one kind has with LTCC
Directly in conjunction with the endogenous protein of passage, when calmodulin is combined with passage, LTCC electric currents presentation high current, the enhancing inactivated by force
State.But under usual state, LTCC passages one of carbon tip (C-terminal) has the ability with calmodulin competition binding passage, leads
Cause passage not combined with calmodulin fully, therefore the enhanced situation that high current inactivates by force can not be showed.Based on above-mentioned competitiveness
Binding isotherm, inventor have separately designed activator and inhibitor.Specifically, when the medicine of design can specificity and passage carbon
When terminal specificity combines, it may be such that passage one of carbon tip fails, passage is more combined with calmodulin, strong so as to which high current be presented
The enhanced situation (being referred to as C-termial Mediated enhanced-mode, CME pattern) of inactivation, this medicine effect pair
Should be in agonist effect.Corresponding, when the medicine of design is the active domain of passage one of carbon tip in itself, medicine can then exercise logical
Road one of carbon tip ability so that calmodulin loses the ability combined with passage, so that the suppression of the weak inactivation of low current is presented in passage
State (being referred to as C-terminal Mediated Inhibition-mode, CMI pattern) processed, drug effect correspond to inhibitor effect (figure
3.a).Further, the regulation and control of LTCC passages, neuronal excitation-transcription can be influenceed and coupled, such as significant pCREB letters
Number path, and then play the effect of regulation and control neuronal development.
Therefore, in one aspect of the invention, the present invention proposes purposes of the polypeptide in medicine is prepared.According to the present invention
Embodiment, the medicine is used to regulate and control L-type valtage-gated calcium channel, and the polypeptide has SEQ ID NO：Any one of 1~8 institute
The amino acid sequence shown.Inventor find, polypeptide of the invention can specific regulatory control LTCC passages, it is emerging so as to influence neuron
Put forth energy-transcribe to couple, such as significant pCREB signal paths, further function as the effect of regulation and control neuronal development.
The amino acid sequence of table 1
According to an embodiment of the invention, purposes of the aforementioned polypeptides in medicine is prepared can also further have following additional
Technical characteristic：
According to an embodiment of the invention, the medicine is used to activate the L-type valtage-gated calcium channel, and the polypeptide has
SEQ ID NO：Amino acid sequence shown in 1~4 any one.
SEQ ID NO：Amino acid sequence shown in 1 is eCaMp's (enhance of CaM pre-association)
Ordered sequence, come from CaV1.3 passage IQ domains.Inventor has found that the polypeptide can make neuron after by electro photoluminescence or chemical stimulation
Cause film potential to rise (neuronal excitation), and then open LTCC passages, Ca2+ oscillations are flowed into neuron by LTCC passages, are swashed
The calmodulin living combined in advance with LTCC passages, and the specific activation CaMKII near passway, the CaMKII after activation enter
Cause gene transcription factor CREB phosphorylation (being referred to as CaMKII-pCREB signal paths) in nucleus, activating ELK 1 B simultaneously influences
The regulation and control of gene transcription level, and then promote neuronal development.
Further, inventor has found, SEQ ID NO：2~4 and SEQ ID NO：Amino acid sequence shown in 1 has height
Homology is spent, and there is similar function, LTCC passages can be activated, so as to promote neuronal excitation-transcription to couple, enter one
Step plays the effect for promoting neuronal development.
According to an embodiment of the invention, half effect concentration of the polypeptide is 6.6 μM.Inventor is to polypeptide pair under various concentrations
The action degree of LTCC paths is studied, and the half effect concentration for obtaining polypeptide is 6.6 μM, that is, produces the medicine of 50% ceiling effect
Concentration is 6.6 μM.
According to an embodiment of the invention, the medicine is by promoting cyclic adenosine monophosphate response element binding protein
Expression, activate pCREB signal paths.Inventor has found that the medicine can raise the combination of phosphorylation CAMP response element
The expression of albumen (pCREB), so as to activate pCREB signal paths, and then promote neuronal development.
According to an embodiment of the invention, the medicine is used to suppress the L-type valtage-gated calcium channel, and the polypeptide has
SEQ ID NO：Amino acid sequence shown in 5~8 any one.
SEQ ID NO：Amino acid sequence shown in 5 is iCaMp's (inhibit of CaM pre-association)
Ordered sequence, come from CaVThe DCRD domains of 1.4 passages.The polypeptide can exercise passage one of carbon tip ability so that calmodulin lose with
The ability that passage combines, so that the holddown of the weak inactivation of low current is presented in passage, so as to suppress neuronal excitation-transcription
Couple, and then suppress neuronal development.
Further, inventor has found, SEQ ID NO：6~8 and SEQ ID NO：Amino acid sequence shown in 5 has height
Homology is spent, and there is similar function, can suppress LTCC passages, be coupled so as to suppress neuronal excitation-transcription, enter one
Step plays the effect for suppressing neuronal development.
According to an embodiment of the invention, the medicine is by suppressing cyclic adenosine monophosphate response element binding protein
Expression, suppress pCREB signal paths.Inventor has found that the medicine can lower the combination of phosphorylation CAMP response element
The expression of albumen (pCREB), so as to suppress pCREB signal paths, and then suppress neuronal development.
According to an embodiment of the invention, the polypeptide is further containing fluorescence labeling sequence and auxiliary cross-film sequence.Fluorescence
The presence of flag sequence is easy to observe medicine and studied.Auxiliary cross-film sequence can realize the cross-film effect of medicine, make
It efficiently plays drug effect.
According to an embodiment of the invention, the fluorescence labeling sequence is the sequence of coding fluorescein isothiocynate；It is described auxiliary
Help cross-film sequence that there is SEQ ID NO：Amino acid sequence shown in 9, it is specific as follows：
RKKRRQRRR(SEQ ID NO：9)
According to an embodiment of the invention, the medicine is used to treat sacred disease and cardiovascular and cerebrovascular disease.Invention human hair
It is existing, polypeptide of the invention can specific regulatory control LTCC passages, coupled so as to influence neuronal excitation-transcription, such as significant
PCREB signal paths, further function as regulation and control neuronal development effect, sacred disease and heart and brain blood can be effectively treated
Pipe disease.
It should be noted that term " treatment ", which is used to refer to, obtains desired pharmacology and/or physiologic effect.The effect
Can be preventative for complete or partial prevention disease or its symptom, and/or just partially or completely cure disease and/or
Can be curative for ill-effect caused by disease." treatment " used herein covers mammal, particularly people's
Disease, including：(a) it is easy ill but not yet make a definite diagnosis prevention disease in the individual fallen ill (such as prevention sacred disease and
Cardiovascular and cerebrovascular disease) or illness generation；(b) disease, such as retardance disease development are suppressed；Or (c) alleviate disease, such as mitigate with
The related symptom of disease." treatment " used herein, which is covered, gives medicine or compound to individual treating, curing, alleviating, changing
Any medication that is kind, mitigating or suppress individual disease, including but not limited to gives the medicine containing this paper to individual in need.
In another aspect of this invention, the present invention proposes a kind of method for screening medicine.According to an embodiment of the invention,
Methods described includes：By Candidate Agents and cells contacting, and whether L-type valtage-gated calcium channel is adjusted before and after detecting cells contacting
Control；And after the contact, the L-type valtage-gated calcium channel is adjusted the finger for being the Candidate Agents as the medicine
Show.Thus, the medicine obtained using the method for screening medicine according to embodiments of the present invention can must effectively regulate and control L-type voltage
Gated calcium channel.
According to an embodiment of the invention, the medicine is polypeptide as previously described institute in the purposes in preparing medicine
Limit.
According to an embodiment of the invention, after the contact, the J of cellCaValue, SCaValue and GCaValue becomes big, is the candidate
Instruction of the medicament as the medicine for activating the L-type valtage-gated calcium channel, after the contact, the J of cellCaValue, SCaValue and GCa
Value diminishes, and is instruction of the Candidate Agents as the medicine for suppressing the L-type valtage-gated calcium channel.
Inventor's discovery, SEQ ID NO：Amino acid sequence shown in 1~4 any one can specific activation LTCC lead to
Road so that passage has big and rapidly calcium current enters, and is presented as JCaValue, SCaValue and GCaValue becomes big.Thus, waited by determining
Before and after selecting medicament and cells contacting, JCaValue, SCaValue and GCaThe change of value, if JCaValue, SCaValue and GCaValue becomes big, then shows to wait
Medicament is selected to couple so as to regulate and control excitement-transcription, further promote as the medicine for activating the L-type valtage-gated calcium channel
Enter neuronal development.
SEQ ID NO：Amino acid sequence shown in 5~8 any one specific can suppress LTCC passages so that passage calcium
Influx is reduced, and is presented as JCaValue, SCaValue and GCaValue diminishes.Thus, before and after by determining Candidate Agents and cells contacting,
JCaValue, SCaValue and GCaThe change of value, if JCaValue, SCaValue and GCaValue diminishes, then shows that Candidate Agents can be used as and suppress institute
The medicine of L-type valtage-gated calcium channel is stated, is coupled so as to regulate and control excitement-transcription, further suppresses neuronal development.
It should be noted that " JCaValue " refers to calcium current density (unit pA/pF), is calcium peak point current (unit pA)
With the ratio of cell size (unit pF), value is bigger, and to represent calcium peak point current bigger；“SCaValue " refers to that calcium relies on inactivation intensity,
SCa=1-I50/Ipeak, wherein I50For 50 milliseconds of calcium current sizes, IpeakFor peak value calcium current size, SCaValue is bigger represent calcium according to
Rely inactivation intensity stronger；“GCaValue " refers to the ratio of peak point current and 300 milliseconds of equilibrium state electric currents, and value is bigger to represent channel peak
Electric current is stronger to the amplifying power of equilibrium state electric current.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Substantially and it is readily appreciated that, wherein：
Fig. 1 shows influences of the activator BayK8644 to mouse cortex neuronal development form and pCREB signal paths,
Wherein, a：The experiment process that BayK8644 is stimulated；b：1 μM of BayK8644 develops under the stimulation of different durations to cortical neuron
The influence of form；c：Influences of the BayK8644 under different stimulated duration to cortical neuron pCREB；
Fig. 2 shows inhibitor isradipine to mouse cortex neuronal development form and the shadow of pCREB signal paths
Ring, wherein, a：The experiment process that isradipine is stimulated；b：5 μM of isradipine are under the stimulation of different durations to cortex neural
The influence of first developmental morphology；c：Influences of the isradipine under different stimulated duration to cortical neuron pCREB；
Fig. 3 shows eCaMp and iCaMp design principle and in HEK293 cells to CaVThe gate characteristic of 1.3 passages
Influence, wherein, a：ICaMp and eCaMp design principle and effective coverage displaying；b：ICaMp and eCaMp is in HEK293 cells
In to CaVThe influence of 1.3 electric currents gate characteristic and GCaStatistical chart, wherein top half are calcium electricity of each group passage under -10mV
Flow diagram, a length of 300ms during opening；The latter half is each group passage GCaCurve；c：Utilize FRET technology for detection iCaMp and right
Answer the binding ability of mutant (V41A) and passage；d：Utilize FRET technology for detection eCaMp original forms ([- IQ-]V) and correspondingly
Mutant (F13A) and the binding ability of passage one of carbon tip；
Fig. 4 shows eCaMp and iCaMp in HEK293 cells to CaVThe parameter system that 1.3 passages gate characteristic influences
Meter, wherein, a：Ca under the influence of eCaMp and iCaMpV1.3 passages open the average calcium current figure of duration in -10mV, 300ms；b：
Ca under the influence of iCaMp_mut, iCaMp, control, CaMp and eCaMp_mutV1.3 channel current schematic diagrames and SCa、JCa、JBa、J300
Statistical chart, the first behavior each group channel current schematic diagram, wherein passage calcium current shows according to the consistent visual effect of peak values
Show, pillar is engineer's scale on the right side of each map of current, and engineer's scale size is 5pA/pF, and passage barium electric current is as shown in pointing to arrow, peak value
Normalized is done according to corresponding group calcium current peak value；Second row SCaThe statistical parameter of inactivation is relied on for passage calcium；The third line and
Fourth line is the peak point current statistical chart of passage calcium current and passage barium electric current；Fifth line is that channel balance state electric current is (corresponding logical
Open electric current during 300ms, J in road300) statistical chart；
Fig. 5 shows activator BayK8644 and inhibitor isradipine in HEK293 cells to CaV1.3 electric current doors
The influence of characteristic is controlled, wherein, a：1 μM of BayK8644 is to CaVThe influence of 1.3 passages；b：5 μM of isradipine are to CaVIt is 1.3 logical
The influence in road；
Fig. 6 shows the eCaMp of chemical synthesis to Ca in HEK293 cellsV1.3 calcium currents gate the influence of characteristic, its
In, a：ECaMp and eCaMp_mut are to Ca under various concentrationsV1.3 under -10mV the influence of calcium current schematic diagram, each group calcium electricity
Flow diagram unanimously does normalized according to equilibrium state；b：The G of eCaMp synthesis polypeptidesCa、JCa、SCaDose-effect curve；c：
300nM and 10 μM of lower eCaMp and eCaMp_mut GCa、JCa、SCaStatistical chart；
Fig. 7 shows iCaMp and eCaMp in HEK293 cells to CaV1.2 passages gate the influence of characteristic, wherein, a：
ICaMp and eCaMp are to CaVThe schematic diagram and G that 1.2 passages (+10mV electric currents) workCaStatistical chart；b-c：CaV1.2 passages by
S after iCaMp and eCaMp regulation and controlCa(b)、JCaAnd JBa(c) statistical chart；
Fig. 8 shows iCaMp and eCaMp to CaVThe influence of 2.1 passages, wherein, a：ICaMp and eCaMp are to CaV1.3、
CaV2.1、CaV2.2 and CaV2.3 SCaInfluence；b：ICaMp and eCaMp are to CaV2.1 pass effects current diagram (+
10mV), calcium relies on inactivation SCaStatistical chart and calcium current size JCaStatistical chart；
Fig. 9 shows iCaMp and eCaMp to CaVThe influence of 2.2 passages, wherein, a：Current diagram (+10mV)；b：Calcium
Rely on inactivation SCaStatistical chart；c：Calcium current size JCaStatistical chart；
Figure 10 shows iCaMp and eCaMp to CaVThe influence of 2.3 passages, wherein, a：Current diagram (+10mV)；b：
Calcium relies on inactivation SCaStatistical chart；c：Calcium current size JCaStatistical chart；
Figure 11 shows iCaMp and eCaMp to mouse cortex neuron CaVThe regulating and controlling effect of 1.3 electric currents；
Figure 12 shows the adjustment effect of iCaMp and eCaMp to cortical neuron pCREB signals；Wherein, a：iCaMp_
The influence of mut, iCaMp, control, eCaMp and eCaMp_mut to cortical neuron ground state pCREB signals, left figure top are experiment
Flow chart, left figure lower section is followed successively by the pCREB levels that the neuron profile of YFP marks and pCREB specific antibodies are shown, right
Figure is the statistics of five groups of neurons, and homogenization processing is done according to the average value of control group；b：ICaMp and iCaMp_mut exist
40mM potassium stimulates the adjustment effect of lower pCREB signals；c：The tune of eCaMp and eCaMp_mut pCREB signals under the stimulation of 20mM potassium
Section acts on；
Figure 13 shows the influence that eCaMp and iCaMp develop to cortical neuron, exists wherein upper figure is five groups of neurons
The neuron morphology schematic diagram that original image and hand drawing under YFP passages go out；It is the total projection length of neuron on the left of figure below
Statistical chart；It is neuron sholl analytic statistics figures on the right side of figure below, sholl analyses are analysis neurite complexity analyzing, are drawn
Radius is the incremental concentric circles of 10m, and the crossing number for calculating concentric circles and projection draws obtained figure；
Figure 14 shows that eCaMp has certain therapeutic effect to the A β 25-35 neurons treated, wherein, A β 25-35
For negative control group, without obvious detrimental effect, A β 25-35 it is positive controls to neuron, there is conspicuousness damage to neuron
Evil effect, A β 25-35 and eCaMp are treatment group, and neuron dendron total length obtains a certain degree of recovery, and upper figure is three groups of god
Aspect graph through first original graph and manual delineation, figure below are projection total length statistical chart and sholl analytic statistics figures；And
Figure 15 shows the polypeptide IQ of the same clan with eCaMpS、IQCAnd IQFAnd the polypeptide DCRD of the same clan with iCaMpS、DCRDC
And DCRDDTo the action effect of LTCC passages, wherein, a：Polypeptide IQ of the same clan eCaMpS、IQCAnd IQFEffect；b：ICaMp is same
The polypeptide DCRD of raceS、DCRDCAnd DCRDDEffect；c：GCaStatistical chart.
Embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carried out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
Embodiment 1
ECaMp and iCaMp N-terminal is fitted together to yellow fluorescence protein YFP in order to observe the expression in cell, with
(iCaMp_V41A, i.e. iCaMp single-point are dashed forward by eCaMp_mut (eCaMp_F13A, i.e. eCaMp simple point mutation) and iCaMp_mut
Become) negative control is used as, mutational site is as shown in Fig. 3 .a.Again with a kind of LTCC subtype C aV1.3 type calcium channels transfect together
Into HEK293 cells, HEK293 cell itselfs do not have detectable valtage-gated calcium channel, therefore can be as good survey
Try carrier.Ca in HEK293 cells is tested by electro physiology meansVThe electrophysiological characteristics of 1.3 passages can learn whether polypeptide has
Effect.
From the point of view of experimental result, agonist polypeptide eCaMp is to CaV1.3 peak current levels have 2.5 times or so of enhancing effect
Fruit, and negative control group is then without enhancing effect (Fig. 4 .b).ECaMp group electric currents fast deactivation after peak point current is reached, into flat
Weigh state electric current, by after average to multiple electric physiological datas, finding the size and control group size phase of eCaMp equilibrium state electric currents
As (Fig. 4 .a).Therefore, a new constant G has been selectedCa(gain factor in calcium current) value (peak value electricity
The ratio of stream and 300 milliseconds of equilibrium state electric currents, value is bigger, and the channel peak electric current that represents is stronger to the amplifying power of equilibrium state electric current)
To describe magnification ratios of the eCaMp to peak point current.
As can be seen that eCaMp groups are shown to the lifting of the conspicuousness of Ca2+ influx and iCaMp group performance groups show to Ca2+ influx
Work property suppresses, and respective negative control group does not make significant difference then to passage.ECaMp groups GCaCompared with control group GCaIt is obviously improved,
(Fig. 3 .b)；In contrast, inhibitor polypeptide iCaMp is to CaV1.3 peak point currents are inhibited close to 50%, and negative control group is then
Unrestraint effect (Fig. 4 .b), iCaMp cause passage to be constantly in low current state, passage GCaValue is obvious to weaken (Fig. 3 .b).
Inventor have detected eCaMp and iCaMp working mechanism using FRET technology (FRET) simultaneously,
ICaMp binding abilities Kd (being similar to the equilibrium constant of chemical balance, smaller to represent binding ability stronger) is 2979, and contrast is negative
Control group iCaMp_V41A Kd values 8015, show that iCaMp can be effectively combined passage IQv domains, and corresponding mutant knot
Conjunction ability is obvious weaker (Fig. 3 .c)；ECaMp is used as FRET pairs using original form IQv domains (Fig. 3 .d), eCaMp original forms pair
The Kd answered is 4538, and contrast negative control group eCaMp_F13A Kd is 10104, and it is good to show that it has with channel C end DCRD
Binding ability, and corresponding mutant binding ability is substantially insufficient, has confirmed the principle using competitive binding.
It can be inferred that eCaMp, which can increase calcium channel, enters calcium ability, passage is possessed journey in short-term and flow into a large amount of calcium letters
Number ability；ICaMp can weaken calcium channel and enter calcium ability, passage is continuously in low current state.
Embodiment 2
In this embodiment, verify traditional activator BayK8644 and inhibitor isradipine to passage calcium current door
Control the influence of characteristic.Test the two kinds of drug concentrations used in document respectively by way of extracellular dosing, respectively 1 μM
BayK8644 and 5 μM of isradipine.As a result show 1 μM of BayK8644 to CaVCalcium current peak value has 3 under 1.3 passage -10mV
Lifting effect again, but conspicuousness suppresses passage SCa, to passage GCaWithout lifting effect.5 μM of isradipine are to CaVIt is 1.3 logical
Calcium current peak value has 74% inhibition, but conspicuousness lifting passage G under road -10mVCa, to passage SCaUnrestraint effect is small
Molecules like pharmaceuticals show enough drug effects.But inventor is changing electric current it is also recognized that for BayK8644 activators simultaneously
While size, important parameter --- the S of inactivation is relied on passage calciumCaIt also result in a certain degree of weakening；And for
Isradipine inhibitor, while size of current is suppressed, to GCaInfluence is also result in, causes GCaIncrease (Fig. 5).It is right
For LTCC, CDI powers may directly influence its development to neuron, have relevant disease such as timothy at present and integrate
There is similar electric current increase in sign (Timothy Syndrome) etc. but inactivation slows down and finally causes what neuronal development was damaged
Situation, from side illustration also there is certain complexity in influences of the BayK8644 and isradipine to neuron.
Embodiment 3
For convenience of using eCaMp and iCaMp this kind of LTCC specific regulatory controls polypeptide, particularly agonist polypeptide eCaMp,
Inventor has synthesized this section of polypeptide using the scheme of chemical synthesis, and the polypeptide group after synthesis turns into FITC-TAT-eCaMp, wherein
FITC (fluorescein isothiocynate) is fluorescence labeling, and TAT is auxiliary cross-film sequence, and eCaMp is 20 amino acid to work.Use
Ca is transfectedVPolypeptide, to ensure that polypeptide can fully work, is directly added into glass by the HEK293 of 1.3 passages as test object
In electrode in liquid, when glass electrode and cell formation high resistance seals and further rupture of membranes formation circuit loop, polypeptide directly enters
Enter in endochylema, the loss of concentration will not be caused.Based on above-mentioned experiment, polypeptide is tested under various concentrations to CaV1.3 action journey
Degree, with GCaValue changes, SCaChange and JCaChange depicts IC50 curves respectively.ECaMp fails to show under low concentration (300nM)
Reveal regulating effect, and under high concentration (10 μM), then good regulating effect is shown, eCaMp_mut is in low concentration and height
Regulating effect can not be shown under concentration, it is final to determine that half effect concentration is respectively 6.6 μM (Fig. 6).
Embodiment 4
The broad spectrum activity to be worked for checking eCaMp and iCaMp to LTCC, while another is demonstrated in nerve, heart etc.
The subtype C a of high expression in multisystemV1.2.Checking understands eCaMp and iCaMp to Ca in HEK293 cellsV1.2 hypotypes are same
Work.Specifically, as shown in Figure 7, eCaMp is effectively increased passage Ca2+ influx, increase passage GCaValue, increase region gray area institute
Show；ICaMp effectively weakens passage Ca2+ influx, reduces passage GCaValue, reduce region as shown in gray area.
The specificity to be worked for research eCaMp and iCaMp to LTCC, inventor demonstrate another in nervous system
Great expression, and often play another large class valtage-gated calcium channel --- the Ca of similar actionV2 passages.CaV2 passages share three
Kind hypotype, respectively N-type, P/Q types and R types, correspond to Ca respectivelyV2.1、CaV2.2 and CaV2.3.Inventor is in HEK293 cells
The influence of eCaMp and iCaMp to the gate characteristic of these three hypotypes is examined, finds iCaMp and eCaMp to CaV1.3 play it is double
To regulating and controlling effect, and to all CaV2 hypotypes without significant regulating effect, can not change CaV2 passage calcium current sizes
(JCa) and inactivation power (SCa) (Fig. 8, Fig. 9 and Figure 10), therefore effects of the explainable eCaMp and iCaMp to LTCC is with higher
Specificity.
Embodiment 5
ECaMp and iCaMp are have detected in HEK293 Heterologous Systems to CaVAfter 1 regulating and controlling effect, inventor further examines
Survey eCaMp and iCaMp and give birth to Ca in sourceV1 system --- work ability in neuron, takes neonate ICR mice cortical neuron
Culture transfected eCaMp and iCaMp and continued culture to the 11st day, with electro physiology in cortical neuron respectively to the 9th day
CaV1.3 passage electrophysiological characteristicses are recorded, to ensure that the electric current of record is CaV1.3 calcium current electric currents, inventor are configured with spy
Different recording solution (includes CaV2 inhibitor MVIIC and GVIA, and 1 μM of certain density LTCC inhibitor
Nimodipine, it can thoroughly suppress Ca under the solubilityV1.2 passages and the Ca for retaining larger compositionV1.3).In the recording solution
Under recorded eCaMp and iCaMp to neuron CaV1.3 calcium currents also have and Ca in HEK293 cellsV1.3 calcium currents are consistent
Regulating effect (Figure 11).ECaMp effectively increases passage Ca2+ influx level, to passage GCa、JCaAnd SCaThree important gates
Parameter with the having conspicuousness effect of raising, and iCaMp serves opposite regulating and controlling effect, while three groups of data can also keep flat
The uniformity of weighing apparatus state electric current.
Embodiment 6
Understood based on above-mentioned electric physiological data, the regulation and control of eCaMp and iCaMp to LTCC electric currents, its mechanism of action and regulation and control
As a result huge difference be present with traditional activator (BayK8644) and inhibitor (isradipine).Inventor's design
ECaMp, adjust and cause that passage has big and rapidly calcium current enters that (eCaMp shows as J after calcium channelCaBecome big, SCaBecome big and GCa
Become big, and BayK8644 shows as JCaBecome big, SCaDiminish, GCaIt is constant), it is more beneficial for starting neuronal development signal path
(CaMKII-pCREB signal paths), it is expected to turn into the medicine for promoting neuronal development.ICaMp adjusts passage small and flat to enter
(iCaMp shows as J to calcium modeCaDiminish, SCaDiminish and GCaDiminish, and isradipine shows as JCaDiminish, SCaIt is constant, GCa
Become big), it is unfavorable for starting developmental character signal path, is expected to turn into the medicine for suppressing neuronal development.For checking inventor's design
Medicine drug effect, inventor selected neonate ICR mice cortical neuron as research object, eCaMp and iCaMp distinguished
Transfection, which enters, has cultivated in 5 days mouse cortex neurons, continue culture observe two days later neuron pCREB signal paths it is strong and weak and
Neuron morphology.Result of study finds that the neuron for having transfected eCaMp groups is higher by than control group neuron pCREB signal intensities
About half, and the basic indifference of negative control group.Correspond, iCaMp groups neuron is than control group neuron pCREB signals
Intensity it is weak about half, and the basic indifference of negative control group.Under the stimulation of high potassium, eCaMp has more than control group to pCREB
Strong amplification effect, and iCaMp can not cause larger pCREB signals than control group substantially.Therefore inventor it is known that
ECaMp has good facilitation effect to neuron pCREB signal paths, and iCaMp has to neuron pCREB signal paths
Good inhibition (Figure 12).
Further, inventor is also detected to neuron morphology, the results showed that, eCaMp group neurons make neuron
Faster, dendron length is longer for development, negative control group and control group indifference.ICaMp group neurons then inhibit neuron to send out
Educate, dendron length shortens, and negative control group and control group indifference (Figure 13).Therefore inventor it could be assumed that, eCaMp can
Promote cortical neuron development, iCaMp can suppress neuronal development.
Embodiment 7
In this embodiment, therapeutic actions of the eCaMp for neuron under pathological conditions is tested, in some sacred diseases
In such as Alzheimer's disease (also referred to as senile dementia), the pathological state degenerated with apoptosis can be presented in neuron, and its pathology shows
As first it is A β abnormal cleavages and aggregation, therefore medically often using A β 25-35 processing neurons to simulate under Alzheimer's disease
Neuron, use this method and transfect eCaMp into neuron to observe eCaMp to the god under this kind of pathological conditions
There is therapeutic effect through member, A β 35-25 are negative control group, and to neuron without obvious detrimental effect, A β 25-35 are positive control
Group, there is conspicuousness to damage effect to neuron, A β 25-35 and eCaMp are treatment group, and neuron dendron total length obtains necessarily
The recovery (Figure 14) of degree.As a result showing, neure damage can effectively be suppressed really by having transfected the neuron of eCaMp groups,
It can expect that eCaMp is expected to play certain therapeutic effect in treatment neurotrosis or sacred disease.
Embodiment 8
Figure 15 shows the polypeptide IQ of the same clan with eCaMpS、IQCAnd IQFAnd the polypeptide DCRD of the same clan with iCaMpS、DCRDC
And DCRDDTo the action effect of LTCC passages.ICaMp active domains are DCRDF, extension of the same clan includes DCRDS、DCRDCAnd DCRDD
There is the conservative of height, and four kinds of DCRD are respectively provided with the effect for suppressing passage Ca2+ influx, show as reducing passage GCa。eCaMp
Active domain is IQD, extension of the same clan includes IQS、IQCAnd IQF, it may have it is well-conserved, and it is respectively provided with the effect for promoting channel opener
Fruit, show as increasing passage GCa。
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the different embodiments or example and the feature of different embodiments or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (10)
1. purposes of the polypeptide in medicine is prepared, it is characterised in that the medicine is used to regulate and control L-type valtage-gated calcium channel, institute
Stating polypeptide has SEQ ID NO：Amino acid sequence shown in 1~8 any one.
2. purposes according to claim 1, it is characterised in that the medicine leads to for activating the valtage-gated calcium of the L-type
Road, the polypeptide have SEQ ID NO：Amino acid sequence shown in 1~4 any one.
3. purposes according to claim 2, it is characterised in that half effect concentration of the polypeptide is 6.6 μM.
4. purposes according to claim 2, it is characterised in that the medicine is by promoting phosphorylation CAMP to react
The expression of element conjugated protein, activate pCREB signal paths.
5. purposes according to claim 1, it is characterised in that the medicine leads to for suppressing the valtage-gated calcium of the L-type
Road, the polypeptide have SEQ ID NO：Amino acid sequence shown in 5~8 any one.
6. purposes according to claim 5, it is characterised in that the medicine is reacted by suppressing phosphorylation CAMP
The expression of element conjugated protein, suppress pCREB signal paths.
7. purposes according to claim 1, it is characterised in that the polypeptide further contains fluorescence labeling sequence and auxiliary
Cross-film sequence,
Preferably, the fluorescence labeling sequence is the sequence of coding fluorescein isothiocynate；
The auxiliary cross-film sequence has SEQ ID NO：Amino acid sequence shown in 9.
8. purposes according to claim 1, it is characterised in that the medicine is used to treat sacred disease and cardiovascular and cerebrovascular
Disease.
A kind of 9. method for screening medicine, it is characterised in that including：
By Candidate Agents and cells contacting, and whether L-type valtage-gated calcium channel is adjusted before and after detecting the cells contacting；With
And
After the contact, the L-type valtage-gated calcium channel is adjusted the instruction for being the Candidate Agents as the medicine.
10. according to the method for claim 9, it is characterised in that after the contact, the J of cellCaValue, SCaValue and GCaValue is equal
Become big, be instruction of the Candidate Agents as the medicine for activating the L-type valtage-gated calcium channel,
After the contact, the J of cellCaValue, SCaValue and GCaValue diminishes, and is that the Candidate Agents are used as the suppression L-type voltage
The instruction of the medicine of gated calcium channel.
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| CN101686952A (en) * | 2007-03-12 | 2010-03-31 | Vm生物医药公司 | Novel agents of calcium ion channel modulators |
| WO2010105856A1 (en) * | 2009-03-19 | 2010-09-23 | Consiglio Nazionale Delle Ricerche | Methods and compositions for modulating cardiac contractility |
| CN102149327A (en) * | 2008-07-11 | 2011-08-10 | Sru生物系统公司 | Methods for identifying modulators of ion channels |
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| CN101686952A (en) * | 2007-03-12 | 2010-03-31 | Vm生物医药公司 | Novel agents of calcium ion channel modulators |
| CN102149327A (en) * | 2008-07-11 | 2011-08-10 | Sru生物系统公司 | Methods for identifying modulators of ion channels |
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| CN114250238A (en) * | 2021-11-26 | 2022-03-29 | 北京航空航天大学 | Gene-encoded neuron development regulation polypeptide and application thereof |
| CN114250238B (en) * | 2021-11-26 | 2023-08-25 | 北京航空航天大学 | Gene-encoded neuron development regulatory polypeptide and application thereof |
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