Conformational Dynamics and Regulatory Mechanisms in the KCNH Family of Ion Channels

SUMMARY The KCNH family of potassium channels comprise the ether á go-go related gene (ERG) channels, ether á go-go (EAG) channels and ether á go-go like (ELK) channels. In response to changes in transmembrane voltage, KCNH channels undergo opening and closing conformational changes to control the movement of potassium ions across the cell membrane and dictate cell excitability. But fundamental questions have not been answered as to the basic mechanisms of voltage-dependent gating. In this proposal we will examine the KCNH channel voltage sensor domain (VSD) and the positively charged S4 domain helix and measure how these domains dynamically rearrange during channel open and closing (gating). Guided by static structures, we will use non-canonical amino acid (ncAA) technology to incorporate photoactivatable or fluorescent amino acids into the S4 of KCNH channels. We will utilize ncAA technology with simultaneous electrophysiological recordings to investigate channel function and photoactivation or fluorescence measurements to measure channel structural changes. The results of our proposed experiments will show the dynamics of the fundamental S4 motions that are a hallmark of voltage-dependent gating in KCNH (and other) channels. KCNH channels contain novel intracellular domains, including an N-terminal Per-Arnt-Sim (PAS) domain and a C-terminal Cyclic Nucleotide Binding Homology Domain (CNBHD). We discovered that the PAS and CNBHD directly interact in hERG channels to regulate gating. Here we show that the PAS and CNBHD move relative to each other during channel gating, and we will leverage ncAA methods to examine how conformational changes at the PAS-CNBHD interface regulate voltage-dependent gating. Distinct from other KCNHs, hERG is of extraordinary physiological importance. Two hERG isoforms (hERG1a and hERG1b) co-assemble to form a native channel (IKr) that repolarizes the human cardiac action potential (AP). Genetic mutations in hERG and inhibition of hERG channels by the off-target action of drugs reduce hERG currents, leading to inherited and acquired forms of long QT syndrome (LQTS), a predisposition to cardiac arrhythmias and sudden death. Recently, we identified a TRIO and f-actin binding protein (TRIOBP-1) that binds the hERG C-terminus and discovered that a hERG1a/hERG1b/TRIOBP-1 complex had a functional interaction with the voltage-gated calcium channel that was cardioprotective in stem cell-derived cardiomyocytes. Here we will use a toolbox of methods to ascertain key interactions among the players in this macromolecular complex and how they regulate the cardiac action potential. Completion of these studies will lead to a mechanistic understanding of how angstrom scale dynamics underlie gating in KCNH channels and how the mechanisms and interactions in a macromolecular complex shape the cardiac AP. Finally, we will identify how LQT2 mutants perturb physiological and biophysical mechanisms in hERG channels, which will lead to personalized diagnosis and treatments for human diseases.