Voitenko S, Purnyn S, Omeltchenko I, Dyadyusha G G, Zhorov B, Brovtsina N, Skok V
Bogomoletz Institute of Physiology, Kiev, USSR.
Mol Pharmacol. 1991 Aug;40(2):180-5.
The effects of (+)-sparteine, a ganglionic blocking agent, on acetylcholine (ACh)-induced membrane currents and on fast excitatory postsynaptic currents (EPSCs) were studied in the neurons of rat isolated superior cervical ganglion, with the whole-cell patch-clamp recording method and the two-electrode voltage-clamp method, respectively. (+)-Sparteine (2 microM) reduced the ACh-induced current caused by activation of nicotinic ACh receptors (AChRs) in a voltage-independent manner at membrane potentials of -50 mV to +30 mV, whereas its blocking effect increased at more negative membrane potentials. The dose-response relationship for ACh was modified by 2 microM (+)-sparteine at -50 mV and at -90 mV in a fashion typical for competitive rather than noncompetitive antagonists. The apparent mean open time of the AChR channel, as estimated from the power density spectrum of the ACh-induced current fluctuations at -90 mV, was not decreased by 2 microM (+)-sparteine, in contrast to what was observed with hexamethonium, the well known open-channel blocker for ganglionic AChRs. At higher concentrations, i.e., 5 microM and 10 microM (lower concentrations were not effective), (+)-sparteine reduced the amplitude of the EPSC and the time constant of the EPSC decay. The former effect was voltage independent, whereas the latter effect was voltage independent at membrane potentials of -70 mV and more positive and increased at membrane potentials of -90 and -110 mV. These results suggest that (+)-sparteine produces in ganglionic AChRs a competitive blocking effect and, in addition, an open-channel blockade. The latter component probably provides a smaller contribution than does the former to the blockade by (+)-sparteine of the ACh-induced current. Conformational analysis of the (+)-sparteine molecule was performed, and the dimensions of the molecule were measured. Minimum dimensions of the space-filling profile for two conformers, high and low populated, were found to be 7.3 x 7.9 A and 6.8 x 7.5 A, respectively. Both profiles are larger than the channel profile at which the open-channel blockers have been suggested to bind, which may explain comparatively low open-channel-blocking activity of (+)-sparteine.
采用全细胞膜片钳记录法和双电极电压钳法，分别研究了神经节阻断剂（+）-鹰爪豆碱对大鼠离体颈上神经节神经元中乙酰胆碱（ACh）诱导的膜电流和快速兴奋性突触后电流（EPSCs）的影响。（+）-鹰爪豆碱（2μM）在膜电位为-50mV至+30mV时，以电压非依赖性方式降低了由烟碱型ACh受体（AChRs）激活引起的ACh诱导电流，而其阻断作用在更负的膜电位时增强。在-50mV和-90mV时，2μM（+）-鹰爪豆碱以竞争性而非非竞争性拮抗剂典型的方式改变了ACh的剂量-反应关系。根据-90mV时ACh诱导电流波动的功率密度谱估计，AChR通道的表观平均开放时间并未因2μM（+）-鹰爪豆碱而降低，这与神经节AChRs的著名开放通道阻断剂六甲铵的情况相反。在较高浓度下，即5μM和10μM（较低浓度无效），（+）-鹰爪豆碱降低了EPSC的幅度和EPSC衰减的时间常数。前一种效应是电压非依赖性的，而后一种效应在膜电位为-70mV及更正时是电压非依赖性的，在膜电位为-90mV和-110mV时增强。这些结果表明，（+）-鹰爪豆碱在神经节AChRs中产生竞争性阻断作用，此外还产生开放通道阻断作用。后一种成分对（+）-鹰爪豆碱阻断ACh诱导电流的贡献可能比前一种成分小。对（+）-鹰爪豆碱分子进行了构象分析，并测量了分子尺寸。发现两种构象（高丰度和低丰度）的空间填充轮廓的最小尺寸分别为7.3×7.9Å和6.8×7.5Å。这两种轮廓都大于已提出开放通道阻断剂结合的通道轮廓，这可能解释了（+）-鹰爪豆碱相对较低的开放通道阻断活性。
