Mapping of the structure - function role of important selected ion channels for normal physiology

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The overarching theme of my research is to characterize the structure-function relationships responsible for the distinctly different physiologic characteristics of HCN and hERG (KCNH2) ion channels. We take advantage of the fact that they have domains of distinct amino acid sequence similarities and differences. Our experimental approach links in silico models of the tertiary structure of the channels and their chimeras to patch clamp assessment of function. The purpose is to understand the structural determinants of differences in their permeation paths, ion selectivity and gating characteristics. For potassium channels, the pore-lining sequence (S/T)(V/I)G(Y/F)G plays a pivot role in K+-selectivity. The Backx laboratory examined the impact of mutation substitutions near the selectivity filter of HCN4 (CIGYG) by reverting it to that seen in potassium channels (SIGYG). Their findings indicated that mutations of the selectivity filter of HCN4 to the SIGYG K+ channel sequence was insufficient to confer K+ selectivity to HCN4. They concluded that differences in selectivity of HCN4 versus K+ channels originated from differences outside the P-loop. The gating characteristics of HCN and hERG are also distinctly different. HCN encodes a channel that is activated at hyperpolarizing potentials and induces an inward depolarizing current, whereas the hERG current is activated by depolarized potentials and induces an outward repolarizing current. There has been an explosive growth of structural and functional information about ion channels. Intriguingly, recent reports show that a mutant (S4-R1-Ser) in the voltage-sensor transmembrane segments of the Shaker K+ channel supports an alternative ion-permeation pathway via the voltage-sensor segments without flux through the conventional selectivity filter. These permeation pathways are termed “omega” currents. We discovered that a single point mutation in the voltage-sensor of hERG (K525S (Ser)) generates a dofetilide-insensitive inward current elicited by hyperpolarization with features quite similar to wild-type HCN4. Even so, there are unresolved differences between K525S hERG and HCN4 including lithium selectivity and pharmacologic block of HCN4 by µMol cesium. Most importantly, we only have circumstantial evidence that HCN is a consequence of an omega current. Our intent is to link atomistic models to patch clamp studies to provide an in-depth understand the topologic rearrangements generating omega currents and their physiologic relevance to HCN4 currents. Experimentally our approach uses in silico designed chimeras, and phylogenetic approach to structure-function. The emerging role of omega currents in normal physiology is potential important to regulate action potential shape, automaticity and synchronicity.