Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239.
Proc Natl Acad Sci U S A. 2013 Oct 29;110(44):17886-91. doi: 10.1073/pnas.1314356110. Epub 2013 Oct 15.
K(+) channels distinguish K(+) from Na(+) in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K(+) binding at the S2 site. We also show that an ester substitution at the S2 site in the KvAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K(+) channels in K(+) compared with Rb(+) are due to different ion occupancies at the S2 site.
钾离子通道（K(+) channels）在选择性滤器中区分钾离子（K(+)）和钠离子（Na(+)），选择性滤器由四个离子结合位点（S1-S4，位于细胞外到细胞内）组成，主要由蛋白质骨架中的羰基氧构建而成。除了离子鉴别外，选择性滤器还通过一种称为 C 型失活的门控过程调节离子穿过膜的流动。C 型失活的一个特征是依赖于可渗透离子，但可渗透离子调节 C 型失活的机制尚不清楚。为了研究这一点，我们使用选择性滤器蛋白质骨架中的酰胺到酯取代来改变特定位点的离子结合，并确定对失活的影响。蛋白质骨架中的酰胺到酯取代是通过蛋白质半合成或体内无义抑制方法引入的。我们表明，KcsA 通道中 S1 位的酯取代不会影响失活，而 S2 和 S3 位的酯取代则显著降低失活。我们确定了 KcsA S2 酯突变体的结构，发现酯取代消除了 S2 位的钾离子结合。我们还表明，KvAP 通道中 S2 位的酯取代具有类似的减缓失活的作用。我们的结果将 C 型失活与 S2 位的离子占据联系起来。此外，它们表明 K(+)通道与 Rb(+)相比失活的差异是由于 S2 位的离子占据不同。
