Characterization of cardiac IKs channel gating using voltage clamp fluorometry

Abstract

Voltage-gated ion channels make up a superfamily of membrane proteins involved in selectively or non-selectively conducting charged ions, which can carry current in and out of cells, in response to changes in membrane voltage. Currents carried by ion channels influence the voltage across the cell membrane, which can trigger changes in the conductance of neighboring voltage-gated channels. In this way, signals, measured as transient changes in voltage called action potentials, can be sent through and between cells in order to transmit information quickly and efficiently throughout excitable systems. My thesis work focuses on elucidating the mechanisms underlying the voltage-dependent gating of a member of the voltage gated potassium (Kv) channel family, KCNQ1 (Kv7.1). Like other members of the voltage gated potassium family, the KCNQ1 channel is made up of four subunits, each containing a voltage sensing domain and a pore-forming domain. Tetrameric channels form with a single central pore domain, and four structurally independent voltage sensing domains. KCNQ1 plays roles both in maintenance of the membrane potential (it forms a leak current in epithelial cells throughout the body) as well as a very important role in resting membrane potential reestablishment (it forms a slowly activating current important in action potential repolarization in cardiac cells). In order to serve these varied functions, KCNQ1 displays uniquely flexible gating properties among Kv channels. Evidence of this flexibility is found in the observation that the presence or absence of various beta subunits can cause the channel to be non-conducting, slowly activating with a large conductance, quickly activating with a small conductance, or constitutively active. My thesis project has been to unravel the mechanisms underlying these very different phenotypes, focusing on the role of the voltage sensor and its coupling to the channel gate. Most of this work focuses on the role of KCNQ1 in the heart, where it comprises the alpha subunit of the slowly activating delayed rectifier current, IKs. This current plays a major role in repolarization of the cardiac action potential, evidenced in part by its major role in shortening the action potential in the face sympathetic stimulation, which leads to phosphorylation-induced increase in IKs current. Further evidence for the importance of IKs to proper cardiac function is found through the identification of many mutations to IKs that result in cardiac arrhythmia, most notably Long QT syndrome, which results from loss of IKs current and an associated prolongation of the cardiac action potential. In addition, gain-of-function IKs mutations have been implicated in Short QT Syndrome and an inherited form of atrial fibrillation. In order to understand mechanisms underlying the physiological and pathophysiological functions of IKs, a more complete picture of its structure and function are needed. One major goal in the pursuit of a more complete characterization of IKs is to understand the interaction between the…