Functional roles of charged residues in the putative voltage sensor of theHCN2 pacemaker channel

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contrib ute to pacemaking activity in specialized neurons and cardiac myocytes, HCN channels have a structure similar to voltage-gated K+ channels but have a much larger putative S4 transmembrane domain and open in response to membra ne hyperpolarization instead of depolarization. As an initial attempt to de fine the structural basis of HCN channel gating, we have characterized the functional roles of the charged residues in the S2, S3, and S4 transmembran e domains. The nine basic residues and a single Ser in S4 were mutated indi vidually to Gin, and the function of mutant channels was analyzed in Xenopu s oocytes using two-microelectrode voltage clamp techniques, Surface membra ne expression of hemagglutinin-epitope-tagged channel proteins was examined by chemiluminescence. Our results suggest that 1) Lys-291, Arg-294, Arg-29 7, and Arg-300 contribute to the voltage dependence of gating but not to ch annel folding or trafficking to the surface membrane; 2) Lys-303 and Ser-30 6 are essential for gating, but not for channel folding/trafficking; 3) Arg -312 is important for folding but not gating; and 4) Arg-309, Arg-315, and Arg-318 are crucial for normal protein folding/trafficking and may charge-p air with Asp residues located in the S2 and S3 domains.