An Analysis of the Variations in Potency of Grayanotoxin Analogues in Modifying Frog Sodium Channels of Differing Subtype

采用膜片钳方法，观察了蛙背根神经节细胞和蛙心脏Na+通道上河豚毒素敏感(河豚毒素-S)和不敏感(河豚毒素-I)钠离子通道对谷氨酰胺毒素类似物GTX-I和α-二氢GTX-II的反应。只有当重复的去极化脉冲先于单个测试去极化时，GTX诱发的修饰才会发生；在测试脉冲期间，修饰表现为峰值Na~+电流的降低并伴随着持续的Na~+电流。通过将通过GTX修饰的Na+通道的持续电流的电导与通过未修饰的Na+通道的控制峰电流的电导归一化来量化GTX对全细胞Na+电流的修饰。通过绘制归一化电导与GTX浓度的关系图，建立了GTX修饰的钠离子通道的剂量-反应关系。至于背根神经节细胞中的河豚毒素-I钠离子通道，GTX-I的EC50和最大归一化电导分别为35mM和0.23，α-二氢GTX-II的EC50和最大归一化电导分别为54mM和0.37。在细胞贴壁膜片上记录通过单个Na+通道的电流，以阐明GTX类似物在修饰DRO TTX-I通道和心室Na+通道时效力显著不同的原因。在背根神经节细胞和心室肌细胞中，经两个GTX类似物修饰的单个Na+通道具有相似的相对电导(范围：0.25-0.42)和开放通道概率(范围：0.5-0.71)。根据这些观察，我们得出结论，DRG TTX-I和心室全细胞Na~+电流对所研究的GTX类似物的反应性的差异与被修饰的Na~+通道的数量有关。

Responses of TTX-sensitive (TTX-s) and insensitive (TTX-i) Na^+ channels, in frog dorsal root ganglion (DRG) cells and frog heart Na^+ channels, to two grayanotoxin (GTX) analogues, GTX-I and alpha-dihydro-GTX-II, were examined, using the patch clamp method. GTX-evoked modification occurred only when repetitive depolarizing pulses preceded a single test depolarization ; modification, during the test pulse, was manifested by a decrease in peak Na^+ current accompanied by a sustained Na^+ current. GTX-evoked modification of whole-cell Na^+ currents was quantified by normalizing the conductance for sustained currents through GTX-modified Na^+ channels to that for the control peak current through unmodified Na^+ channels. The dose-response relation for GTX-modified Na^+ channels was constructed by plotting the normalized conductance against GTX concentration. With respect to TTX-i Na^+ channels found in DRG cells, the EC_<50> (helf-maximal concentration) and maximal normalized conductance were estimated to be 35 mu M and 0.23 for GTX-I, and 54 mu M and 0.37 for alpha-dihydro-GTX-II.By contrast, TTX-s Na^+ channels in DRG, and Na^+ channels in ventricular cells were found to have a much lower sensitivity to both GTX analogues. Currents through single Na^+ channels were recorded from cell-attached membrane patches to elucidate, the factors responsible for the marked differences in potency of the GTX analogues in modifying DRO TTX-i and ventricular Na^+ channels. In DRG cells and ventricular myocytes, single Na^+ channels modified by the two GTX analogues (both at 100 mu M), had similar relative conductances (range : 0.25-0.42) and open channel probabilities (range : 0.5-0.71). From these observations, we conclude that the differences in responsiveness of DRG TTX-i, and ventricular whole cell Na^+ currents to the GTX analogues studied are related to the number of Na^+ channels modified.