Wang Guangyu, Covarrubias Manuel
Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107, USA.
J Gen Physiol. 2006 Apr;127(4):391-400. doi: 10.1085/jgp.200509442. Epub 2006 Mar 13.
The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a "hanging gondola" below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1-S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn(2+) site thiolate groups in the T1-T1 interface; therefore, we concluded that the T1-T1 interface is functionally active and not protected by Zn(2+) (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55-69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (k(MTSET)) is dramatically enhanced in the activated state relative to the resting and inactivated states (approximately 260- and approximately 47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, k(MTSET) is steeply voltage dependent in a manner that is precisely correlated with the peak conductance-voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn(2+) site. Furthermore, cross-linking of cysteine pairs across the T1-T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex voltage-dependent gating rearrangements of eukaryotic Kv channels are not limited to the membrane-spanning core but must include the intracellular T1-T1 interface. Oxidative stress in excitable tissues may perturb this interface to modulate Kv4 channel function.
大多数真核生物电压门控钾通道（Kv通道）的细胞内四聚化结构域（T1）以“悬挂式吊篮”的形式存在于跨膜区域下方，该跨膜区域通过S4电压传感器与主要S6门之间的机电耦合直接控制激活门控。然而，关于T1结构域对Kv通道门控的假定贡献，人们了解得要少得多。这种可能性在机制上很有趣，因为T1-S1连接子将T1结构域连接到电压传感结构域。此前，我们证明硫醇特异性试剂通过以状态依赖的方式与T1-T1界面中的天然Zn(2+)位点硫醇盐基团反应来抑制Kv4.1通道；因此，我们得出结论，T1-T1界面具有功能活性，且不受Zn(2+)保护（Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55-69）。在此，我们共表达Kv4.1通道和辅助亚基（KChIP-1和DPPX-S），以研究MTSET对T1结构域中三个界面半胱氨酸的可及性的状态和电压依赖性。结果表明，相对于静息状态和失活状态，激活状态下的平均MTSET修饰速率常数（k(MTSET)）显著提高（分别约为260倍和约47倍）。至关重要的是，在产生不同激活曲线的三种不同条件下，k(MTSET)呈陡峭的电压依赖性，其方式与峰值电导-电压关系精确相关。这些观察结果强烈表明，Kv4通道门控与亚基间Zn(2+)位点天然T1半胱氨酸的电压依赖性可及性变化紧密耦合。此外，T1-T1界面上半胱氨酸对的交联诱导了通道的显著抑制，这支持了T1在通道门控中的功能动态作用。因此，我们得出结论，真核生物Kv通道复杂的电压依赖性门控重排不仅限于跨膜核心，还必须包括细胞内T1-T1界面。可兴奋组织中的氧化应激可能会扰乱该界面，从而调节Kv4通道功能。
