Mechanisms of Mode Shift and Gating Polarity in Voltage-dependent Ion Channels
Author | : John Cowgill |
Publisher | : |
Total Pages | : 0 |
Release | : 2020 |
Genre | : |
ISBN | : |
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Electrical signaling is one of the most essential processes to the survival of higher organisms. This is mediated by the regulated flow of ions through the cell membrane catalyzed by a diverse set of membrane proteins known as ion channels. An important channel type opens and closes in response to changes in the membrane potential. These voltage-gated ion channels (VGICs) thus regulate the very stimulus which governs their activity, enabling them to generate and regulate electrical excitability in cells. In this thesis, I examine the structural and thermodynamic aspects of voltage-dependent regulation of channel function. In Chapter Three, I used a hierarchical approach based on recently-solved cryoEM structures of two VGICs to examine the role of various structural elements in voltage sensing, opening the pore, and coupling these processes. I localized discrete elements in hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels that are responsible for conferring an inverted gating response in these channels compared to virtually all other VGICs which activate on depolarization. Surprisingly, the HCN voltage sensor can gate the same pore open in both hyperpolarizing and depolarizing directions suggesting that these channels use a unique voltage sensing mechanism. In Chapter Four, long-timescale molecular dynamics simulations from collaborators revealed a new atomic model of voltage sensing in HCN channels that sheds light on the mechanism of inverted gating polarity. We experimentally validated this novel mechanism using cysteine accessibility measurements and utilized our bipolar constructs to demonstrate that this mechanism underlies the inverted gating polarity of HCN channels. Finally, in Chapter Five I examined the origin mode shift in VGIC gating, a widely observed phenomenon whereby longterm changes in membrane potential alter gating properties. By improving the protocols for recording gating currents, I demonstrated that the hysteresis in gating charge-voltage relationship that is commonly attributed to mode shift stems from non-equilibrium measurement conditions. Throughout these works, my findings are discussed in terms of structural, functional, and thermodynamic implications.