Stotz S C, Haynes L W
Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Alberta, Canada.
Biophys J. 1996 Dec;71(6):3136-47. doi: 10.1016/S0006-3495(96)79507-3.
Tetraalkylammonium compounds and other organic cations were used to probe the structure of the internal and external mouths of the pore of cGMP-gated cation channels from rod and cone photoreceptors. Both rod and cone channels were blocked by tetramethyl- through tetrapentylammonium from the intracellular side in a voltage-dependent fashion at millimolar to micromolar concentrations. The dissociation constant at 0 mV (KD(O)) decreased monotonically with increasing carbon chain length from approximately 80 mM (TMA) to approximately 80 microM (TPeA), where the dissociation constant in rod channels is approximately 50% that of cone channels. N-Methyl-D-glucamine and the buffer Tris also blocked the cone channel in a voltage-dependent fashion at millimolar concentrations, but with lower affinity than similarly sized tetraalkylammonium blockers. Block by tetrahexylammonium (THxA) was voltage-independent, suggesting that the diameter of the intracellular mouth of these channels is less than the size of THxA but larger than TPeA. The location of the binding site for intracellular blockers was approximately 40% across the voltage-drop from the intracellular side. The addition of one carbon to each of the alkyl side chains increased the binding energy by approximately 4 kJ mol-1, consistent with hydrophobic interactions between the blocker and the pore. Cone, but not rod, channels were blocked by millimolar concentrations of extracellular TMA. The location of the extracellular binding site was approximately 13% of the voltage drop from the extracellular side. In cone channels, the two blocker binding sites flank the location of the cation binding site proposed previously.
四烷基铵化合物和其他有机阳离子被用于探究视杆和视锥光感受器中cGMP门控阳离子通道孔的内外口结构。在毫摩尔至微摩尔浓度下，从细胞内侧，四甲基铵至四戊基铵均以电压依赖性方式阻断视杆和视锥通道。在0 mV时的解离常数（KD(O)）随着碳链长度增加而单调降低，从约80 mM（TMA）降至约80 μM（TPeA），其中视杆通道中的解离常数约为视锥通道的50%。N-甲基-D-葡糖胺和缓冲剂Tris在毫摩尔浓度下也以电压依赖性方式阻断视锥通道，但亲和力低于类似大小的四烷基铵阻断剂。四己基铵（THxA）的阻断不依赖电压，表明这些通道细胞内口的直径小于THxA但大于TPeA。细胞内阻断剂结合位点的位置约在从细胞内侧算起跨电压降的40%处。每条烷基侧链增加一个碳原子使结合能增加约4 kJ mol-1，这与阻断剂和孔之间的疏水相互作用一致。毫摩尔浓度的细胞外TMA可阻断视锥通道，但不阻断视杆通道。细胞外结合位点的位置约在从细胞外侧算起跨电压降的13%处。在视锥通道中，两个阻断剂结合位点位于先前提出的阳离子结合位点两侧。
