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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.
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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.
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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.
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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.
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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.