Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel.
Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute 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 membrane hyperpolarization instead of depolarization. As an initial attempt to define the structural basis of HCN channel gating, we have characterized the functional roles of the charged residues in the S2, S3, and S4 transmembrane domains. The nine basic residues and a single Ser in S4 were mutated individually to Gln, and the function of mutant channels was analyzed in Xenopus oocytes using two-microelectrode voltage clamp techniques. Surface membrane expression of hemagglutinin-epitope-tagged channel proteins was examined by chemiluminescence. Our results suggest that 1) Lys-291, Arg-294, Arg-297, and Arg-300 contribute to the voltage dependence of gating but not to channel folding or trafficking to the surface membrane; 2) Lys-303 and Ser-306 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-pair with Asp residues located in the S2 and S3 domains.
Chen
J
J
Department of Medicine, Division of Cardiology and Eccles Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah 84112-5330, USA.
Mitcheson
J S
JS
Lin
M
M
Sanguinetti
M C
MC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D004553:Electric Conductivity; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008163:Luminescent Measurements; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017354:Point Mutation / Q000235:genetics; D015221:Potassium Channels; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D055672:Static Electricity; D014982:Xenopus laevis
C578710:HCN2 protein, human; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D008274:Magnesium