Immke D, Wood M, Kiss L, Korn S J
Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269, USA.
J Gen Physiol. 1999 Jun;113(6):819-36. doi: 10.1085/jgp.113.6.819.
The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = approximately 265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30-100 microM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.
电压门控钾通道Kv2.1在无钾的情况下能传导钠离子。外部的四乙铵（TEAo）以5 mM的半数抑制浓度（IC50）阻断通过Kv2.1的钾电流，但在无钾时则完全无效。加入低浓度的[K+]后，TEAo的阻断作用可被逆转。这表明Kv2.1孔道在外腔经历了阳离子依赖性的构象重排。外腔中赖氨酸（Lys）356和382分别突变为甘氨酸和缬氨酸，使TEAo阻断钾电流的效力提高了半个对数单位。位于外腔外边缘的Lys 356突变在无钾时显著恢复了TEAo的阻断作用（IC50 = 21 mM）。相反，位于靠近TEA结合位点的外腔中的Lys 382突变在无钾时导致TEAo的阻断作用非常弱（推算的IC50约为265 mM）。这些数据表明，导致TEA效力丧失的孔道构象的阳离子依赖性改变延伸到了外腔的外边缘，并且主要涉及Lys 356或传导途径中附近氨基酸的重新定位。内部TEA的阻断在无钾时也完全消失，并且可以用低浓度的[K+]逆转。内部和外部TEA的效力都因阻断通过该通道的钠电流的相同低浓度[K+]（30 - 100 microM）而增加。此外，结合内部和外部TEA阻断的实验表明，钾的作用位点在内部和外部TEA结合位点之间。这些数据表明，选择性过滤器内部也发生了钾依赖性的构象变化，并且内部和外部的构象重排都是由选择性过滤器中钾占据情况的差异引起的。Kv2.1的失活速率依赖于钾，并且与TEAo的效力相关；随着[K+]升高，TEAo变得更有效，失活也变得更快。TEAo的效力和失活速率在相同的[K+]下达到饱和。这些结果表明，Kv2.1中缓慢失活的速率受到构象重排的影响，这些构象重排要么发生在选择性过滤器内部，要么发生在外腔外边缘附近，而这些构象重排与TEA效力的差异有关。
