Department of Physiology and Biophysics, University of Miami Miller School of Medicine, FL 33136, USA.
J Gen Physiol. 2011 Jun;137(6):533-48. doi: 10.1085/jgp.201110616. Epub 2011 May 16.
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels have the largest conductance (250-300 pS) of all K(+)-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA(-) increased currents and MTSET(+) decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high K(+). Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.
大电导钙激活钾（BK）通道是所有钾选择性通道中电导最大的（250-300pS）。然而，离子传导途径的各个部分对电导的贡献尚不清楚。在这里，我们研究了内腔入口对大电导的贡献。四个α亚基上的 E321/E324 残基环绕着内腔入口。为了确定 321/324 是否可从内导通路进入，我们测量了 E321C 和 E324C 通道胞内侧暴露和洗涤巯基试剂前后的单通道电流幅度。MPA(-) 增加了电流，MTSET(+) 减少了电流，321 和 324 之间没有差异，表明 321/324 的侧链可从内导通路进入，对电导有等效影响。对于中性氨基酸，用 321/324 处的大侧链氨基酸取代，减小内腔入口的大小，会降低外向单通道电导，而用较小侧链取代增大入口大小则几乎没有影响。高[K+]（i）可消除外向电导的降低。取代对内向电导影响不大。用一个由串联的一个可变电阻和一个固定电阻组成的模型拟合电导与侧链体积的关系图表明，野生型通道内腔入口的有效直径和长度分别为 17.7 和 5.6Å，入口电阻约占总传导途径电阻的 7%。估计的尺寸与 BK 通道的古细菌同源物 MthK 的结构一致。我们的观察表明，BK 通道具有低电阻、大的内腔入口，入口的大小足以不限制电流，但不会太大。
