Chepilko S, Zhou H, Sackin H, Palmer L G
Department of Physiology and Biophysics, Cornell University Medical College, New York, New York 10021.
Am J Physiol. 1995 Feb;268(2 Pt 1):C389-401. doi: 10.1152/ajpcell.1995.268.2.C389.
The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.
肾钾通道（ROMK2）在非洲爪蟾卵母细胞中表达，采用膜片钳技术评估其传导和门控特性。在浴槽和电极内均含110 mM钾离子的细胞贴附式膜片中，反转电位接近零，内向电导（36 pS）大于外向电导（17 pS）。在切除的内面向外膜片中，当细胞质侧存在5 mM镁离子时通道表现出整流特性，而在无镁离子溶液中则无此现象。当电极内的钾离子被铷离子、铵离子或铊离子（Tl+）取代时也观察到内向电流。在这些条件下的反转电位产生的选择性序列为Tl+ > K+ > Rb+ > NH4+。另一方面，内向电流的斜率电导产生的选择性序列为K+ = NH4+ > Tl+ > Rb+。这两个序列的差异可以通过通道内存在阳离子结合位点来解释，该位点与铷离子和铊离子的相互作用比与钾离子更强，与铵离子的相互作用比与钾离子更弱。另外两种离子，钡离子（Ba2+）和铯离子（Cs+）从外部阻断该通道。1 mM钡离子的作用是降低通道的开放概率，而10 mM铯离子降低表观单通道电流。两种阻断剂的作用都因膜超极化而增强。还在细胞贴附式膜片中研究了通道的动力学。电极内为钾离子时，开放时间的分布可用单指数函数描述（τ0 = 25 ms），而关闭时间的分布需要用两个指数函数描述（τ1 = 1 ms，τ2 = 30 ms）。卵母细胞膜超极化降低开放概率和τ0，并增加τ1、τ2以及长关闭的次数。电极内存在铊离子会显著改变动力学，降低τ0并消除长寿命关闭。这些结果表明通道的门控可能取决于孔道内离子的性质。
