Prod'hom B, Pietrobon D, Hess P
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
J Gen Physiol. 1989 Jul;94(1):23-42. doi: 10.1085/jgp.94.1.23.
We further investigated the rapid fluctuations between two different conductance levels promoted by protons when monovalent ions carry current through single L-type Ca channels. We tested for voltage dependence of the proton-induced current fluctuations and for accessibility of the protonation site from both sides of the membrane patch. The results strongly suggest an extracellular location of the protonation site. We also studied the dependence of the kinetics of the fluctuations and of the two conductance levels on the concentration of permeant ion and on external ionic strength. We find that saturation curves of channel conductance vs. [K] are similar for the two conductance levels. This provides evidence that protonation does not appreciably change the surface potential near the entry of the permeation pathway. The proton-induced conduction change must therefore result from an indirect interaction between the protonation site and the ion-conducting pathway. Concentration of permeant ion and ionic strength also affect the kinetics of the current fluctuations, in a manner consistent with our previous hypothesis that channel occupancy destabilizes the low conductance channel conformation. We show that the absence of measurable fluctuations with Li and Ba as charge carriers can be explained by significantly higher affinities of these ions for permeation sites. Low concentrations of Li reduce the Na conductance and abbreviate the lifetimes of the low conductance level seen in the presence of Na. We use whole-cell recordings to extrapolate our findings to the physiological conditions of Ca channel permeation and conclude that in the presence of 1.8 mM Ca no proton-induced fluctuations occur between pH 7.5 and 6.5. Finally, we propose a possible physical interpretation of the formal model of the protonation cycle introduced in the companion paper.
当单价离子通过单个L型钙通道传导电流时，我们进一步研究了质子促进的两种不同电导水平之间的快速波动。我们测试了质子诱导电流波动的电压依赖性以及膜片两侧质子化位点的可及性。结果强烈表明质子化位点位于细胞外。我们还研究了波动动力学和两种电导水平对渗透离子浓度和外部离子强度的依赖性。我们发现，两种电导水平下通道电导与[K]的饱和曲线相似。这提供了证据，表明质子化不会明显改变渗透途径入口附近的表面电位。因此，质子诱导的传导变化必定是由质子化位点与离子传导途径之间的间接相互作用引起的。渗透离子浓度和离子强度也会影响电流波动的动力学，这与我们之前的假设一致，即通道占据会使低电导通道构象不稳定。我们表明，以Li和Ba作为电荷载体时没有可测量的波动，可以用这些离子对渗透位点的亲和力显著更高来解释。低浓度的Li会降低Na电导，并缩短在有Na存在时所见低电导水平的寿命。我们使用全细胞记录将我们的发现外推到钙通道渗透的生理条件，并得出结论，在存在1.8 mM Ca的情况下，在pH 7.5至6.5之间不会发生质子诱导的波动。最后，我们对配套论文中引入的质子化循环形式模型提出了一种可能的物理解释。
