Molecular Interactions that Contribute to the Regulation of HCN Channels by KCNE2

Hyperpolarization activated cyclic-nucleotide gated (HCN) channels are the molecular correlate of neuronal and cardiac Ih. The four mammalian isoforms that have been identified (HCN1-4) are widely expressed in the central and peripheral nervous systems as well as cardiac conduction tissue. KCNE2 is the only member of the KCNE protein family known to regulate HCN expression, voltage-sensitivity, and gating kinetics. KCNE2-/- mice have diminished Ih in cortical and hippocampal neurons and increased excitability, consistent with experiments in over-expression systems. Here we examine the molecular details of HCN-KCNE2 complex formation, including subunit stoichiometry, and sites of protein-protein interactions. Single-molecule fluorescence experiments indicate that HCN1 channels interact with WT-KCNE2 with a 4:2 stoichiometry in a concentration independent manner. Some KCNE2 gene variants can alter this stoichiometry and alter HCN1 function, while others have no discernible impact. Using computational approaches, we have identified putative sites of interaction in the HCN1-KCNE2 complex that are being validated experimentally. Further biophysical evidence suggest that KCNE2 alters the regulation of HCN1 channels by other modulators including cAMP, which could have potential implications of its role in neuronal function.