Date of Award
Spring 5-15-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Voltage-gated potassium channels (KV) facilitate cellular excitability essential for cardiac contraction and neuronal spiking. In response to changes in the membrane potential, KV channels conduct transmembrane K+ flux to terminate action potentials. Myriad drugs target KV channels to achieve therapeutic goals such as anti-arrhythmic and anti-convulsant effects. Owing to their physiological significance, KV channels are exquisitely regulated by a cohort of auxiliary proteins and signaling molecules. The KCNQ1 or KV7.1 voltage-gated potassium channel stands out as an archetype in this regard. Modulators such as the KCNE subunit family, membrane lipid PIP2, and calmodulin (CaM) enable KCNQ1 to conduct phenotypically distinct currents in excitable and non-excitable tissues. In the heart, KCNQ1 conducts the voltage-dependent IKs current to shorten action potentials during the fight-or-flight response. In gut epithelia, KCNQ1 passes a voltage-independent K+ leak current to facilitate apical salt secretion. Hence, in-depth understanding of KCNQ1 promises insights into physiology in diverse tissues and novel therapeutic strategies.Normal KCNQ1 functions are produced by modular channel domains, including the voltage-sensing domain (VSD), the ion-permeating pore domain, and the cytosolic carboxy-terminus domain (CTD). During channel activation, KCNQ1 domains engage in three molecular processes: (1) VSD activation, (2) VSD-pore coupling, or interactions between the VSD and the pore, and (3) pore opening. The VSD activates in a two-step manner, from an initial resting state to a stable intermediate state, then to the fully activated state. VSD occupancy of the intermediate and activated states both couple to the pore to trigger K+ conductance – leading to two distinct KCNQ1 open states. The dual open state paradigm in KCNQ1 appears unique among voltage-gated channels. Despite this general understanding, critical details remain unknown. What is the molecular motion underlying VSD activation? What VSD-pore coupling mechanisms enable the two open states? How do CaM, PIP2, and the KCNQ1 CTD figure into the activation process? Lastly, what is the physiological significance of the two KCNQ1 open states?In this dissertation, I address the above questions in collaboration with others to provide a molecularly-detailed framework for voltage-dependent activation of the KCNQ1-CaM channel complex. We begin by explicitly correlating the functional intermediate and activated VSD states to their respective structural states, thereby elucidating the molecular motion associated with KCNQ1 VSD activation. Next, we map the critical VSD-pore coupling residues that enable the intermediate and activated VSD to trigger pore opening, describing a two-stage VSD-pore coupling mechanism that accounts for both open states. We next expand the scope include the KCNQ1 CTD, CaM and PIP2. We find that VSD transition from the intermediate to the activated state triggers a change of interactions between the VSD with PIP2 and with CaM, thereby switching the open state at the pore. The study of the KCNQ1 dual open state gating scheme provides insights into the principles of voltage-dependent gating in general. With statistical evolutionary analysis, we postulate that the KCNQ1 two-stage activation mechanism is broadly conserved in many KV channels. Moreover, I uncover disease-associated CaM variants that may disrupt this process to contribute to arrhythmia, highlighting the physiological significance of CaM in KCNQ1 activation. In all, this work provides molecular details underlying KCNQ1 VSD activation, VSD-pore coupling, and CaM regulation to culminate in a mechanistic framework that explains how the KCNQ1-CaM channel complex generates two open states. Findings in this dissertation further demonstrate that auxiliary subunits regulate the dual open states to enable KCNQ1 to conduct phenotypically distinct currents in diverse tissues, with dysregulation of this process potentially contributing to arrhythmogenesis.
Language
English (en)
Chair
Jonathan R. Silva
Committee Members
Jianmin Cui, Jeanne Nerbonne, Rajan Sah, Andrea Soranno,