Thompson Jill, Begenisich Ted
Department of Pharmacology and Physiology, University of Rochester Medical Center, NY 14642, USA.
J Gen Physiol. 2005 Jun;125(6):619-29. doi: 10.1085/jgp.200509251. Epub 2005 May 16.
We have examined the voltage dependence of external TEA block of Shaker K(+) channels over a range of internal K(+) concentrations from 2 to 135 mM. We found that the concentration dependence of external TEA block in low internal K(+) solutions could not be described by a single TEA binding affinity. The deviation from a single TEA binding isotherm was increased at more depolarized membrane voltages. The data were well described by a two-component binding scheme representing two, relatively stable populations of conducting channels that differ in their affinity for external TEA. The relative proportion of these two populations was not much affected by membrane voltage but did depend on the internal K(+) concentration. Low internal K(+) promoted an increase in the fraction of channels with a low TEA affinity. The voltage dependence of the apparent high-affinity TEA binding constant depended on the internal K(+) concentration, becoming almost voltage independent in 5 mM. The K(+) sensitivity of these low- and high-affinity TEA states suggests that they may represent one- and two-ion occupancy states of the selectivity filter, consistent with recent crystallographic results from the bacterial KcsA K(+) channel. We therefore analyzed these data in terms of such a model and found a large (almost 14-fold) difference between the intrinsic TEA affinity of the one-ion and two-ion modes. According to this analysis, the single ion in the one-ion mode (at 0 mV) prefers the inner end of the selectivity filter twofold more than the outer end. This distribution does not change with internal K(+). The two ions in the two-ion mode prefer to occupy the inner end of the selectivity filter at low K(+), but high internal K(+) promotes increased occupancy of the outer sites. Our analysis further suggests that the four K(+) sites in the selectivity filter are spaced between 20 and 25% of the membrane electric field.
我们研究了在2至135 mM范围内的一系列内部K⁺浓度下，外部TEA对Shaker K⁺通道的阻断作用的电压依赖性。我们发现，在低内部K⁺溶液中，外部TEA阻断的浓度依赖性不能用单一的TEA结合亲和力来描述。在更去极化的膜电压下，与单一TEA结合等温线的偏差增大。这些数据可以用一个双组分结合方案很好地描述，该方案代表了两个对外部TEA亲和力不同的、相对稳定的导电通道群体。这两个群体的相对比例受膜电压的影响不大，但确实取决于内部K⁺浓度。低内部K⁺促进了具有低TEA亲和力的通道比例增加。表观高亲和力TEA结合常数的电压依赖性取决于内部K⁺浓度，在5 mM时几乎与电压无关。这些低亲和力和高亲和力TEA状态的K⁺敏感性表明，它们可能代表了选择性过滤器的单离子和双离子占据状态，这与细菌KcsA K⁺通道最近的晶体学结果一致。因此，我们根据这样一个模型分析了这些数据，发现单离子和双离子模式的固有TEA亲和力之间存在很大（几乎14倍）差异。根据该分析，单离子模式（在0 mV时）中的单个离子在内选择性过滤器末端的偏好是外末端的两倍。这种分布不会随内部K⁺而改变。双离子模式中的两个离子在低K⁺时更喜欢占据选择性过滤器的内末端，但高内部K⁺会促进外位点占据增加。我们的分析进一步表明，选择性过滤器中的四个K⁺位点在膜电场的20%至25%之间间隔分布。
