Pharmacological activities of α3β2 and α3β4 nicotinic acetycholine receptors with different α and β subunit stoichiometries / 中国病理生理杂志

AIM:To compared the differential sensitivity of nicotinic acetycholine receptors (nAChRs) consisting of α and β subunits with different ratios.METHODS:The cRNA of α and β subunits was obtained by in vitro transcription.The α3β2 and α3β4 nAChR subtypes were expressed in Xenopus laevis oocytes by microinjection of cRNA coding α and β subunits at α∶β ratios of 1∶10, 1∶1 and 10∶1.The pharmacological activities of nAChRs to agonist acetycholine (ACh) and antagonist α-conotoxin (CTx) RegⅡA were investigated by two-electrode voltage-clamp techniques.RESULTS:For α3β2 nAChR expressed at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 91.2 μmol/L, 104.4 μmol/L and 130.6 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 40.2 nmol/L, 36.4 nmol/L and 42.3 nmol/L, respectively.For α3β4 nAChR at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 44.0 μmol/L, 110.0 μmol/L and 230.0 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 226.8 nmol/L, 71.5 nmol/L and 49.4 nmol/L, respectively.CONCLUSION:The results imply that the α3 and β4 subunit stoichiometry can change the structure and pharmacological activity of α3β4 nAChR, but the stoichiometry of α3 and β2 subunits has no effect on α3β2 nAChR.

Construction of α3(S147T)* Nicotinic Acethlcholine Receptor Mutants and Study of Their Function / 中国药学杂志

OBJECTIVE: To construct the point mutants of α3* nicotine acetylcholine receptors (nAChRs), optimize the method of receptor mutagenesis and investigate the function of the mutants by using the agonist acetycholine (Ach). METHODS: The α3* nAChRs mutants were constructed by PCR mediated site-directed mutation techniques. Point mutated primers were designed according to rat α3 subunit gene. The cRNA of α3 subunit point mutant was synthesized by in vitro transcription. The expression of mutants in Xenopus oocytes were detected by two-electrode voltage-clamp techniques. Gating properties of the two mutants were detected by Ach. RESULTS: Mutants of α3β2 and α3β4 nAChRs subtypes were constructed successfully. The half effective concentrations (EC50) of wild types α3β2 and α3β4 nAChRs were 55.33 and 163.00 μmol·L-1, respectively. While the EC50 of α3(S147T)β2 and α3(S147T)β4 nAChRs mutants were 33.10 and 121.10 μmol·L-1, respectively. CONCLUSION: The construction of mutation from the 147th serine to threonine of α3 subunit can provide a function model to make more other receptor mutants, and would be helpful to interrogate the interaction between drug and α3* nAChR.

Construction of functional and concatenated dimers of two-pore-domain potassium channel TREK-1 / 军事医学

Objective To explore the feasibility of adding a flexible linker between two-pore-domain potassium channel TREK-1 (TWIK related K + channel 1)monomers to construct a tandem-linked dimer.Methods PCR was used to add a flexible linker between the two TREK-1 monomers.The cRNA obtained from in vitro transcription using the above vector was injected into Xenopus oocytes.After 24 -48 h,currents were recorded from these oocytes using a two-electrode voltage clamp.The effects of extracellular Ba2 + and pH on TdTREK-1 were observed and compared with those of native dimeric TREK-1.Results The tandem-linked dimeric TdTREK-1 was highly expressed in Xenopus oocytes.The currents through these channels were inhibited by extracellular Ba2 +and acidification.Furthermore,the responsiveness of the concatenated dimers to these extracellular stimuli was similar to that of native dimers.Conclusion Adding a flexible linker between the two monomers to construct the tandem-linked dimer does not affect the expression and gating properties of TREK-1, suggesting that the method be feasible.Such a method will allow the manipulation of a single subunit,which will help basis study the structure and function of TREK-1.

Inhibitory effect of lead acetate on TRPA1 channel in mice and humans / 中国药理学与毒理学杂志

OBJECTIVE To investigate the inhibitory effect of lead acetate on transient receptor potential A1(TRPA1)channel. METHODS TRPA1-mediated calcium influx in mice dorsal root ganglion(DRG) neurons and HEK293 cells expressing nouse TRP1 (mTRPA1) and human TRPA1 (hTRPA1) was recorded by intracellular calcium imaging. TRPA1-mediated currents were detected by two-electrode voltage clamp. RESULTS Lead acetate 3.0 and 10.0μmol·L-1 inhibited external calcium influx in DRG neurons by(36.7 ± 4.1)% and(79.4 ± 3.1)%(n=5),respectively. The inhibitory effect of lead acetate on hTRPA1-mediated current was concentration-dependent. Lead acetate 0.3, 1.0, 3.0, 10.0 and 30.0μmol · L-1 inhibited the amplitudes of currents by(1.0 ± 0.7)%,(11.6 ± 0.8)%,(57.7 ± 3.2)%,(93.6 ± 2.6)%and(91.2±2.0)%(n≥4),respectively,with the IC50 2.4μmol·L-1. CONCLUSION TRPA1 channel may be an endogenous target of lead. Lead acetate inhibits TRPA1 channel at a very low concentration.

Effect of XinBao pill on electrophysiological properties of human HCN4 channel / 中国生化药物杂志

Objective To explore the basic medical mechanism of XinBao pill on electrophysiological characteristics of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), and illustrate the mechanism of its therapeutical effect on bradycardia.Methods Human HCN4 mRNA was injected into the Xenopus laevis oocytes, after incubated for 2 ~3 days, channel current properties of HCN4 perfused with 40 mg/L XinBao Pill were observed by double electrode voltage clamp technique.Results At-90mV test potential, compared with control group (no XinBao pill), HCN4 channel peak current and tail current in 40 mg/L XinBao pill group had obvious changes, and V1/2 from ( -103.61 ±3.57)mV to ( -106.42 ±5.33)mV in XinBao pill group, from( -81.11 ±4.26)mV to( -86.36 ±7.44)mV in control group.The values of k from (15.15 ±2.23)mV to (17.33 ±3.58) mV in XinBao pill group, from(11.78 ±0.85)mV to(12.39 ±1.51)mV in control group(n=10).At test potential -90 mV, 40 mg/L XinBao pill perfusion fluid decreased the instantaneous current of(0.15 ±0.24)%, the EC50 was (30.8 ±4.8)mg/L (n=8).At test potential-140 mV~-100 mV level, 40 mg/L XinBao pill group increased the channel activation time constant compared with control group[(226.73 ±31.36)ms vs(143.67 ± 21.44)ms;-140 mV,n=10,P<0.05].40 mg/L XinBao pill group increased the channel deactivation time constant compared with control group [(1293.53 ±95.02)ms vs (647.12 ±61.35)ms;-140 mV,n=10,P<0.05].Conclusion The XinBao pill enhances the instantaneous current of HCN4 in a concentration-dependent manner, and extents channel activation and deactivation processes.

Electrophysiological methods for drug assessment

Background: Drug assessment is an important step to determine the effectiveness and safety of each compound. Any candidate substances are repeatedly tested to examine the activity and working mechanism. Drugs produce various effects through interaction with ionotropic receptors. Electrophysiological methods are used to detect such effects by monitoring the drug-ionotropic receptor interaction. Researchers are now focusing to develop simple and quick methods for drug assessment. Objective: This mini-review presents current drug assessment using electrophysiological methods, and clarifies various problems involved in the assessment.

Regulation of Antiarrhythmic Drug Propafenone Effects on the C-type KV1.4 Potassium Channel by PHo and K+

The effects of the antiarrhythmic drug propafenone at c-type kv1.4 channels in Xenopus laevis oocytes were studied with the two-electrode voltage-clamp techinique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4 delta N channels. During recording, oocytes were continuously perfused with control solution or propafenone. Propafenone decreased the currents during voltage steps. The block was voltage-, use-, and concentration- dependent manners. The block was increased with positive going potentials. The voltage dependence of block could be fitted with the sum of monoexponential and a linear function. Propafenone accelerated the inactivate of current during the voltage step. The concentration of half-maximal block (IC(50)) was 121 micrometer/L. With high, normal, and low extracellular potassium concentrations, the changes of IC(50) value had no significant statistical differences. The block of propafenone was PH- dependent in high-, normal- and low- extracellular potassium concentrations. Acidification of the extracellular solution to PH 6.0 increased the IC50 values to 463 micrometer/L, alkalization to PH 8.0 reduced it to 58 micrometer/L. The results suggest that propafenone blocks the kv1.4 delta N channel in the open state and give some hints for an intracellular site of action.

Design of Voltage-clamp-controlled Current-clamp / 生物化学与生物物理进展

Previous research rcvealed that distortion is detected in transient voltage signal recorded with traditional patch clamp amplifier under current clamp mode, which is essentially resulted by electronic design of the headstage of the patch clamp. A new kind of headstage is designed to modify the defect, the circuit of which not only measures the transient potentials as the classical voltage follower does but also is quite suitable for the standard voltage-clamp mode. Furthermore, the technique of voltage-clamp-controlled current-clamp is applied for modifying the conventional patch-clamp amplifier, the variable low-pass filter is added into the circuit to reduce the response speed of voltage-clamp module, thus the transient potentials changes can be measured while membrane potential is kept at a constant value. Bridge balance circuitry is designed to eliminate the voltage drop while the variable current injected into the electrode. And fast capacitance compensation stage of conventional PCA is modified to nentralize the capacitance and accelerate system response speed for current-clamp mode. The experirnental results on cell model demonstrate that modified PCA meets the rcquirement of monitoring transient potential changes in electrophysiology research.

ENDOMORPHIN-1 IS MORE POTENT THAN ENDOMORPHIN-2 IN INHIBITION OF SYNAPTIC TRANSMISSION IN SUBSTANTIA GELATINOSA OF ADULT RAT SPINAL CORD / 神经解剖学杂志

Effects of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) on synaptic transmission were investigated on neurons in substantia gelatinosa (SG) of the spinal dorsal horn by whole-cell voltage clamp recording. Both EM-1 (1 μmol/L) and EM-2 (1 μmol/L)remarkably reduced the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). These effects were antagonized by 3-funaltrexamine ( β-FNA, 10 μmol/L), a selective μ-opioid receptor antagonist. Noticeably, EM-1 showed higher potency in decreasing the frequency of mEPSCs and mIPSCs than that of EM-2. These results indicate that EMs suppress both excitatory and inhibitory synaptic transmission by activating presynaptic μ-opioid receptors in the SG and EM-1, compared with EM-2, might be a more potent endogenous analgesic at the spinal cord level.

Mechanism for baclofen inhibition on quantal glutamate release in spinal dorsal horn neurons / 中国药理学通报

Aim To investigate the inhibition mechanisms of baclofen, a specific GABA B receptor agonist, on quantal glutamate release in the rat spinal dorsal horn neurons.Methods Whole-cell voltage-clamp technique was performed on dorsal horn neurons in rat spinal cord slice to record glutamatergic spontaneous miniature excitatory postsynaptic currents (mEPSCs). Baclofen action on quantal glutamate release was assessed by analyzing the change of mEPESC to baclofen perfusion.Results Baclofen(10 ?mol?L -1,50 s) depressed the frequency, but not amplitude distribution of glutamatergic mEPSCs, indicating baclofen presynaptic depression on glutamate release. The depression on frequency of mEPSCs persisted in Ca 2+-free solution, or in the presence of K + conductance blocker, 4-AP. On the other hand, the depression was occluded by forskolin, an activator of adenylate cyclase, but not protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). N-ethylmaleimide (NEM), a sulphydryl alkylating agent, which destroys G protein, abolished baclofen depression.Conclusion Not presynaptic K +, Ca 2+ conductance or PKC, but G protein and/or cAMP pathway are involved in the baclofen depression on glutamate release in rat spinal dorsal horn;this depression might contribute to the analgesic action of baclofen at spinal level.

Increase of L-type calcium current by cGMP-dependent protein kinase regulates in rabbit ventricular myocytes

BACKGROUND: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current (ICa) in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of ICa by cGMP. However, there is no direct evidence that cGMP-PK can stimulate ICa in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK an ICa in rabbit ventricular myocytes. METHODS AND RESULTS: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of ICa by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. ICa was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal ICa. cGMP-PK also increased basal ICa. The stimulation of basal ICa by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal ICa by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When ICa was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of ICa. In the presence of cGMP-PK, already increased ICa was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. CONCLUSIONS: We present evidence that cGMP-PK stimulated basal ICa by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.

Characterization of voltage-gated potassium currents in dorsal root ganglion neurons of neonatal rats

Dorsal root ganglion (DRG) is composed of neuronal cell bodies of primary afferents with diverse functions. Various types of ion channels present on DRG neurons may reflect those functions. In the present study, voltage-gated potassium currents in DRG neurons of neonatal rats were characterized by whole-cell voltage clamp method. Two types of delayed rectifier and three types of transient potassium currents were identified according to their electrophysiological properties. The delayed rectifier currents were named IKe (early inactivating) and IKl (late inactivating). Steady state inactivation of IKe began from -100 mV lasting until -20 mV. IKl could be distinguished from IKe by its inactivation voltage range, from -70 mV to + 10 mV. Three transient currents were named IAf (fast inactivation), IAi (intermediate inactivation kinetics), and IAs (slow inactivation). IAf showed fast inactivation with time constant of 10.6 +/- 2.0 msec, IAi of 36.9 +/- 13.9 msec, and IAs of 60.6 +/- 2.9 msec at +30 mV, respectively. They also had distinct steady state inactivation range of each. Each cell expressed diverse combination of potassium-currents. The cells most frequently observed were those which expressed both IKl and IAf, and they had large diameters. The cells expressing IKe and expressing IKe, IAi, and IAs usually had small diameters. Judging from cell diameter, capsaicin sensitivity or action potential duration, candidates for nociceptor were the cells expressing IKe, expressing IKe and IAi, and expressing IKe and IAs. The types and distribution of potassium currents in neonatal rat DRG were similar to those of adult rat DRG (Gold et al, 1996b).

Effect of dipfluzine on hERG potassium currents expressed in xenopus oocytes / 中国药理学通报

Aim To investigate the effect of dipfluzine on hERG potassium currents expressed heterologously in xenopus oocytes.Methods Using Xenopus oocytes expression system,the current amplitude and kinetic characteristics of hERG were measured with the two electrode voltage-clamp technique before and after dipfluzine application.Results Dipfluzine(8 nmol?L-1~5 ?mol?L-1)concentration-dependently inhibited hERG currents;the concentration for half maximal inhibition(IC50)was 98.0 nmol?L-1.Dipfluzine-induced inhibition of hERG currents was voltage dependent at membrane potentials between-10 and 40 mV.Dipfluzine at 1 ?mol?L-1 didn't statistically shifted V1/2 of hERG currents activation.Dipfluzine at 1 ?mol?L-1 significantly decreased the activating time constants and the deactivating time constants,and enhanced hERG currents activation and deactivation.Conclusion Dipfluzine concentration-dependently inhibits hERG currents and modifies kinetic characteristics of hERG activation and deactivation,which may be correlated with its antiarrhythmic effect.