期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：49
页码：15096-15100
DOI：10.1073/pnas.1510526112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe selectivity filter of K+ channels is responsible for their exquisite ion selectivity. This region is also responsible for C-type inactivation, a regulatory process in many voltage-dependent K+ channels. Although the functional properties of inactivated channels have been known for decades, the first potential glimpse of their structure emerged from crystal structures of a constricted selectivity filter in an open channel. However, recent studies challenged the suggestion that the constricted selectivity filter is the inactivated structure, leaving open the question of what the inactivated structure looks like. Here, we provide evidence that the thermodynamic properties of the selectivity filter in an inactivated channel are more similar to properties of the conductive channel rather than the constricted open channel. K+ channels are membrane proteins that selectively conduct K+ ions across lipid bilayers. Many voltage-gated K+ (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K+ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na+ or low [K+]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K+ ion binding to channels with an inactivated or conductive selectivity filter is different from K+ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.
关键词：K+ channel ; ion binding ; inactivation ; isothermal titration calorimetry ; electron paramagnetic resonance spectroscopy
