MOLECULAR MECHANISMS OF PACEMAKER CHANNEL FUNCTION

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DESCRIPTION (Verbatim from Applicant's Abstract): The overall goal of this project is to determine the structural basis for function of Hyperpolarization-activated, Cyclic Nucleotide-gated (HCN) channels. These cation-selective channels conduct the pacemaker current of cardiac myocytes and neurons, and are most closely related to the erg family of K+ channels (e.g., HERG). Together, HCN and HERG channels are important regulators of automaticity in atrial pacemaker cells. However, while HERG (and all other voltage-gated K+ channels) are opened by depolarizataion, HCN channels are opened by hyperpolarization. The molecular mechanism for this difference is unknown. We hypothesize that specific electrostatic and H-bonding interactions between the S4 and other transmembrane domains of HCN facilitate channel opening in response to hyperpolarization, but prevent channel opening in response to depolarization. We further hypothesize that the S4-S5 linker couples the movement of the S4 domains to the activation gate. This hypothesis is based on our discovery that a point mutation (D540K) in the S4-S5 linker of HERG destabilizes the closed state and, similar to HCN, permits channels to open in response to hyperpolarizaiton. Wild-type and mutant HCN and HERG channels will be heterologously expressed in Xenopus oocytes and studied using voltage clamp, substituted cysteine accessibility mutagenesis and biochemical techniques. The specific aims are to determine the structural basis for hyperpolarization-dependent opening of HCN and mutant HERG channels, to characterize movement of the S4 domain of HCN2 in response to membrane depolarization and hyperpolarization, and to determine the role of electrostatic interactions between S4 and other transmembrane domains in the folding and gating of HCN channels. These studies will elucidate the molecular mechanisms for gating of HCN channels and further our understanding of the cardiac pacemaker.