CN105935441A - Application of polypeptide in preparation of TTX-R type sodium channel tool molecule - Google Patents
Application of polypeptide in preparation of TTX-R type sodium channel tool molecule Download PDFInfo
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Abstract
The invention discloses an application of a bioactive polypeptide in preparation of a voltage-gated sodium channel tool reagent-TTX-R type sodium channel inhibitor. The bioactive polypeptide is spider sodium-inhibiting peptide-IX (SPNP-IX); tests verify that the active polypeptide has an obvious inhibitory effect on a TTX-R type voltage-gated sodium channel. In the preparation of the voltage-gated potassium channel subtype tool reagent, the extracellular dosing concentration is made to be 100 nmol/L or 500 nmol/L. The 50% effect dose IC50 of the SPNP-IX is 530 nmol/L. The inhibition of the SPNP-IX on the TTX-R type sodium channel shows time compliance, and the SPNP-IX is a good TTX-R type sodium channel tool reagent.
Description
Technical field
The present invention relates to a kind of active polypeptide purposes in ion channel tool reagent, especially prepare with chemical method
Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX(and is abbreviated as SPNP-IX) preparing the examination of voltage-gated sodium channel instrument
Purposes in the inhibitor of agent-TTX-R type sodium channel.
Background technology
Voltage-gated sodium channel is distributed widely in the excitabilities such as neuron, cardiac muscle, vascular smooth muscle, pancreas, skeletal muscle
In tissue, be a kind of important transmembrane structure albumen, its participate in regulation secretion of neurotransmitter, vasoconstriction, pancreatic secretion and
The various physiological processes such as skeletal muscle irritability, are also simultaneously one of the important targets of medicine effect.Sodium channel is at action electricity
Key effect in the generation of position and communication process, voltage-gated sodium channel becomes the action target of a lot of animals and plants toxin.
The structure-function relationship research of sodium-ion channel has become as international study hotspot.Voltage-gated sodium channel is likely to into
Critical treatment target spot for the disease such as nerve and cardiovascular.The zootoxin of nature be a class have the highest practical value and
The tool reagent of application prospect or drug targeting material, be not only venomous animal and resist the powerful mean of natural enemy, be also to be engaged in god
Through biology and Physiologic Studies, the exploitation of natural original new drug and the valuable materials of protein basic research, also grind simultaneously
Study carefully the uniqueness " molecular probe and decipher " of ion channel.Up to now, from many animals (as scorpion, Aranea, Serpentis and ocean are dynamic
Thing etc.) venom in identified the active component of a lot of batrachotoxin.They are by the diverse location phase with sodium channel
In conjunction with thus cause the dynamic characteristic of passage that corresponding change occurs, as passage de-inactivation, passage mouth, easily change activation,
Inactivation delays.By illustrating the molecular mechanism of these specificity modulator batrachotoxins, except going deep in theory
Speculate outside the functional activity difference between the hypotype of sodium channel and gate active mechanism, it is also possible to for they being developed into the treatment mankind
The new drug of relevant disease provides sufficient theoretical basis and wide application prospect.
Summary of the invention
It is contemplated that in provide a kind of Guangxi tassel hair spider Toxic extraction thing-Aranea press down sodium peptide-IX prepare valtage-gated
The application of tool reagent-TTX-R type sodium channel, sodium channel inhibitor, described Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium
Peptide-IX(is abbreviated as SPNP-IX), its aminoacid sequence is:
NH2-Glu Cys Thr Lys Leu Leu Gly Gly Cys Thr Lys Asp Ser Glu Cys Cys Pro
His Leu Gly Cys Arg Lys Lys Trp Pro Tyr His Cys Gly Trp Asp Gly Thr Phe-
NH2,
It is characterized in that Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX as single effective active component for preparing
TTX-R type sodium channel inhibitor.
Described Aranea presses down between the 2nd cysteine of N end and the 16th cysteine of sodium peptide-IX, the 9th half, N end
Between cystine and the 21st cysteine, between the 15th cysteine of N end and the 29th cysteine, form two sulfur respectively
Key.
This Aranea presses down sodium peptide-IX as single effective active component for preparing TTX-R type sodium channel inhibitor, with cell
The mode of external administration is prepared as TTX-R type sodium channel tool reagent.
Aranea presses down sodium peptide-IX preparing TTX-R type sodium channel tool reagent or during inhibitor, the agent that preparation extracellular is administered
Amount is 100 nmol/L or 500 nmol/L.
Aranea presses down the half useful effect dosage IC of sodium peptide-IX suppression TTX-R type sodium channel50It is 530 nmol/L.
Aranea presses down the sodium peptide-IX suppression presentative time compliance to TTX-R type sodium channel, is a kind of preferably TTX-R type
Sodium channel tool reagent.
Accompanying drawing explanation
Fig. 1 is that 100 nmol/L SPNP-IX are on the impact of TTX-R type sodium channel on rat drg neuron.
Fig. 2 is that 500 nmol/L SPNP-IX are on the impact of TTX-R type sodium channel on rat drg neuron.
Fig. 3 is SPNP-IX m-effect relation curve when acting on rat DRG cell TTX-R sodium channel.
Fig. 4 is concentration-effect relation curve that SPNP-IX acts on rat DRG cell TTX-R sodium channel.
Fig. 5 is the SPNP-IX impact on rat DRG cell TTX-R sodium channel current-voltage relationship.
Fig. 6 is the SPNP-IX impact on rat DRG cell TTX-R sodium channel Conductance-Voltage relation.
Fig. 7 is the SPNP-IX dynamic (dynamical) impact of Steady-state inactivation on rat DRG cell TTX-R sodium channel.
Detailed description of the invention
In order to be better understood from and more fully disclose the present invention, the rat back of the body will be used by extracellular route of administration below
Root neural section cell model illustrates that Aranea presses down the TTX-R type sodium channel inhibitory action of sodium peptide-IX.
1, experiment material and method
The acute isolation of 1.1 dorsal root ganglion neurons (DRG cell) is cultivated
Select birth about 4 weeks, the SD rat of body weight 140～200 g, take out rapidly vertebra also with scissors at disconnected neck after death
It is cut into 2～3 sections, then along the direction vertical with rib plane, canalis spinalis is cut off, and be immersed in the beaker filling a small amount of culture fluid;
Tear the one layer of mucosa being attached on canalis spinalis inwall with tweezers, expose the Dorsal ganglion fiber of the intersection being positioned at canalis spinalis and rib.
In Thoracolumbar disk section, can select in about 18 culture dishs being placed into filling 2 mL culture fluid;Under anatomic microscope, use tip
Tweezers and Wei Nasi cut and isolate neuroganglion.Peel off after wrapping in the floccule outside joint and axon, put into and fill about 0.5 mL cultivation
In the culture dish of liquid.Suck liquid with suction pipe, all neuroganglions of separator well are shredded, the most broken more good.After shredding, proceed to disappear
Change liquid, 34 DEG C, the environment of concussion frequency 110 rpm carries out enzymolysis, digestion and reacts 20 min.Period takes out every 10 min
Inhale with liquid-transfering gun and beat for several times；In Digestive system, add pancreatin inhibitor, terminate enzymolysis.The Cell sap that digestion is obtained by sterile working
Proceed to centrifuge tube is centrifuged (800 rpm, 5 min), remove supernatant, add 8 mL long-term cultivation liquid containing 10% calf serum.
It is divided into 3～4 wares after re-suspended cell, puts into incubator (5%CO2, 95% air) in, 37 DEG C cultivate 3～4 h adherent.
1.2 patch-clamp electrophysiologic activity experiments
Patch clamp experiments is all carried out in room temperature (25 ± 1 DEG C), uses whole-cell patch-clamp recording technique.Select smooth visible, the born of the same parents of plasma membrane
Matter uniform DRG cell or have the HEK293T cell of green fluorescence as experimental cell.Electric current record passes through full cell patch
Tongs technology utilizes EPC9 amplifier (HEKA company, German) to carry out on computers.Computer recording and the system of analysis use
Pulse+Pulsefit 8.0 software.Glass electrode pipe is borosilicate glass capillary tube (Nanjing spring water education experiment equipment
Factory).Glass electrode two step draws and forms, and polished instrument (Narishige, Japan) polishing rear electrode tip diameter is about 3 m,
Charging electrode solution rear electrode resistance is 1-3M Ω.Patch clamp experiments to be carried out at ambient temperature, temperature in whole experimentation
Variation the most up and down less than 2 DEG C.Use SigmaPlot 9.0 software analysis experimental result.
2, experimental result and analysis
2.1 SPNP-IX impact on TTX-R type voltage-gated sodium channel
We have detected the SPNP-IX impact on DRG cell TTX-R type voltage-gated sodium channel.Result as shown in Figure 1, 2,
The SPNP-IX of 100nM and 500 nM has obvious inhibitory action to TTX-R sodium channel, and maximal percentage inhibition is respectively 21 ± 2%
(n=5) and 54 ± 4% (n=4).And, it is different from other spider venom (such as JZTX-I), when SPNP-IX part suppression
After sodium current, the shape of electric current is consistent with comparison current shape, illustrates that they the most substantially change the activation of sodium current and inactivation
Dynamic characteristic, thus it is presumed that toxin binding site on sodium channel is not likely to be site 3.SPNP-IX suppresses TTX-R
Type sodium current speed is the most very fast, has time dependent.When concentration is 10 μMs, can reach within the time of about 2 min
To the suppression degree (Fig. 4) of 100%.The time constant of the SPNP-IX suppression DRG cell TTX-R sodium current of 10 μMs is 21.2 ±
1.7 s (n = 5).And the SPNP-IX of 10 μMs is about 97% to the suppression ratio of the TTX-R sodium channel of tranquillization closed mode,
Show that SPNP-IX all has similar affinity to being in tranquillization closedown with the sodium channel of state of activation.SPNP-IX is to rat DRG
The suppression of cell TTX-R sodium channel current has concentration compliance, their half effective inhibition concentration (IC50) it is 530
Nmol/L (Fig. 4).When dose curve is with Hill formula fitting, can draw the Hill coefficient of concentration-response curve, size is
0.95。
2.2 SPNP-IX impact on TTX-R type voltage-gated sodium channel state of activation
Under whole cell voltage clamp recording pattern, cell is clamped down on when-80 mV, give a series of test voltage respectively, its
Excursion is-80～+60 mV, and stride is+10 mV, and the persistent period is 50 ms, can obtain TTX-R on rat DRG cell
Current-voltage (I-V) graph of relation of sodium channel, and the activation threshold of passage, peak inrush current activation electricity can be disclosed
Pressure and reversal potential size.Under the conditions of blank, when cell membrane potential is clamped down at-80 mV, initiateing of TTX-R sodium channel
Activating voltage and be about-40 mV, maximum current peak activates voltage and is about-10 mV, and reversal potential is about+25 mV.At cell
Around add the SPNP-IX of 500 nM, allow after toxin and cytosis 3 min, then stimulate induction I-V with identical depolarization
Curve, result such as Fig. 5.SPNP-IX can make the initial activation potential of TTX-R and TTX-R sodium channel and peak point current activate voltage
To direction of depolarization drift about+10 mV, but reversal potential change is inconspicuous, illustrates that toxin does not change with the interaction of passage
The selectivity that variable conduit is penetrating to ion.SPNP-IX also has similar impact, energy to the Conductance-Voltage curve of TTX-R sodium channel
Conductance-Voltage (G-S) relation curve of TTX-R sodium channel is caused to drift about about+10 mV(Fig. 6 toward direction of depolarization).
2.3 SPNP-IX impact on TTX-R type voltage-gated sodium channel Steady-state inactivation
We use the dipulse stimulation mode of a standard, have further looked at SPNP-IX and have led to rat DRG cell TTX-R sodium
The dynamic (dynamical) impact of Steady-state inactivation in road.Dipulse stimulus parameter is: first clamped down in advance by cell membrane potential at-120 mV, continues
Time is 1 s, and then transmembrane potential is changed into-80 mV, and the time is 0.5 ms, it is therefore an objective to eliminate the charging shape of cell membrane capacitance
State, then depolarization current potential is transitted to-10 mV, the persistent period is 50 ms, and afterwards, transmembrane potential is returned to-80 mV.Under carrying out
When once stimulating, only need to change pre-Clamping voltages (-120 mV-0 mV, stride is+10 mV), and test voltage level is same
On.What Fig. 7 represented is under different pre-Clamping voltages, institute's induced current and the ratio size of maximum current (i.e.-120 mV).Will
In figure, all groupings of data points Boltzmann equations are fitted, and can draw the half Steady-state inactivation voltage (V of passage1/2) value.
Such as Fig. 7, for TTX-R sodium channel, under collating condition, V1/2For-38.9 ± 1.1 mV(n=7), after adding JZTX-IX, V1/2Become
Become-41.9 ± 1.0 mV(n=6).Result shows that the Steady-state inactivation feature of TTX-R sodium channel is had not significant impact by SPNP-IX,
And the most substantially change the slope of control curve.
SEQUENCE LISTING
<110>Changsha Qin Cai bio tech ltd
<120>Aranea presses down sodium peptide-IX
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 35
<212> PRT
<213> SPNP-IX
<400> 1
Glu Cys Thr Lys Leu Leu Gly Gly Cys Thr Lys Asp Ser Glu Cys
1 5 10 15
Cys Pro His Leu Gly Cys Arg Lys Lys Trp Pro Tyr His Cys Gly
16 20 25 30
Trp Asp Gly Thr Phe
31 35
Claims (7)
1. Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX and is preparing voltage-gated sodium channel tool reagent-TTX-
Application in the inhibitor of R type sodium channel, described Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX(and is abbreviated as SPNP-
IX), its aminoacid sequence is:
NH2-Glu Cys Thr Lys Leu Leu Gly Gly Cys Thr Lys Asp Ser Glu Cys Cys Pro
His Leu Gly Cys Arg Lys Lys Trp Pro Tyr His Cys Gly Trp Asp Gly Thr Phe-
NH2,
It is characterized in that Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX as single effective active component for preparing
TTX-R type sodium channel inhibitor.
A kind of Guangxi the most according to claim 1 tassel hair spider Toxic extraction thing-Aranea press down sodium peptide-IX prepare valtage-gated
Application in the inhibitor of tool reagent-TTX-R type sodium channel, sodium channel, it is characterised in that described Aranea presses down the N of sodium peptide-IX
Hold between the 2nd cysteine and the 16th cysteine, between the 9th cysteine of N end and the 21st cysteine, N end
Disulfide bond is formed respectively between 15th cysteine and the 29th cysteine.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down sodium peptide-IX as single effective active
Component is used for preparing TTX-R type sodium channel inhibitor.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down sodium peptide-IX and is prepared as extracellular administration
TTX-R type sodium channel tool reagent.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down sodium peptide-IX at preparation TTX-R type sodium
When passage tool reagent or inhibitor, the dosage that preparation extracellular is administered is 100 nmol/L or 500 nmol/L.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down the half useful effect agent of sodium peptide-IX
Amount IC50It is 530 nmol/L.
Application process the most according to claim 1 and 2, it is characterised in that it is temporal dependence that Aranea presses down the effect of sodium peptide-IX
's.
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| CN201610234427.7A CN105935441A (en) | 2016-04-17 | 2016-04-17 | Application of polypeptide in preparation of TTX-R type sodium channel tool molecule |
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| CN201610234427.7A CN105935441A (en) | 2016-04-17 | 2016-04-17 | Application of polypeptide in preparation of TTX-R type sodium channel tool molecule |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261117A (en) * | 2017-06-20 | 2017-10-20 | 长沙沁才生物科技有限公司 | Applications of the SPNP 27 in Kv4.3 potassium channel inhibitors are prepared |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872878A (en) * | 2006-06-22 | 2006-12-06 | 湖南师范大学 | Jingzhao toxin V |
| CN105154515A (en) * | 2015-07-08 | 2015-12-16 | 长沙沁才生物科技有限公司 | Application of bioactive peptide in preparation of potassium channel subtype tool reagents |
| CN105148255A (en) * | 2015-07-08 | 2015-12-16 | 长沙沁才生物科技有限公司 | Application of natural peptide in preparation of Kv4.2 potassium channel tool reagents |
-
2016
- 2016-04-17 CN CN201610234427.7A patent/CN105935441A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872878A (en) * | 2006-06-22 | 2006-12-06 | 湖南师范大学 | Jingzhao toxin V |
| CN105154515A (en) * | 2015-07-08 | 2015-12-16 | 长沙沁才生物科技有限公司 | Application of bioactive peptide in preparation of potassium channel subtype tool reagents |
| CN105148255A (en) * | 2015-07-08 | 2015-12-16 | 长沙沁才生物科技有限公司 | Application of natural peptide in preparation of Kv4.2 potassium channel tool reagents |
Non-Patent Citations (1)
| Title |
|---|
| MEICHUN DENG等: ""Jingzhaotoxin-IX, a novel gating modifier of both sodium and potassium channels from Chinese tarantula Chilobrachys jingzhao"", 《NEUROPHARMACOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261117A (en) * | 2017-06-20 | 2017-10-20 | 长沙沁才生物科技有限公司 | Applications of the SPNP 27 in Kv4.3 potassium channel inhibitors are prepared |
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Application publication date: 20160914 |