Department of Physiology and Biophysics, University of Washington, Seattle, WA, USA.
Department of Biochemistry, University of Washington, Seattle, WA, USA.
Nat Commun. 2021 May 14;12(1):2802. doi: 10.1038/s41467-021-23062-7.
Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A' helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that - in contrast to electromechanical coupling for channel activation - the A' helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.
起搏器超极化激活环核苷酸门控 (HCN) 离子通道表现出反向电压依赖性门控，通过膜超极化而不是去极化激活。海胆 HCN (spHCN) 通道也会随着超极化而失活，这种失活仅在没有环核苷酸的情况下发生。在这里，我们应用过渡金属离子 FRET、膜片钳荧光法和 Rosetta 建模来测量 spHCN 通道激活和失活之间结构重排的差异。我们发现，去除 cAMP 会导致 C 接头相对于跨膜结构域产生很大的刚体旋转，使 C 接头的 A' 螺旋与电压感应 S4 螺旋紧密接近。此外，在没有 cAMP 的情况下，超极化会引起 C 接头的旋转，但在存在 cAMP 的情况下则不会。这些结果表明 - 与通道激活的机电偶联相反 - A' 螺旋用于将 S4 螺旋运动偶联到通道失活，这可能是 CNBD 家族通道的一种保守机制。
