title: Molecular basis for the different activation kinetics of the pacemaker channels HCN2 and HCN4.
abstract: The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant (tau act), which is fast for HCN2 (144 ms at -140 mV) and slow for HCN4 (461 ms at -140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased tau act of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted tau act to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in tau act. Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.
authors.affiliation: Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Strasse 29, 80802 Munich, Germany. stieber@ipt.med.tu-muenchen.de
authors.forename: Juliane
authors.initials: J
authors.lastname: Stieber
authors.forename: Anna
authors.initials: A
authors.lastname: Thomer
authors.forename: Barbara
authors.initials: B
authors.lastname: Much
authors.forename: Angela
authors.initials: A
authors.lastname: Schneider
authors.forename: Martin
authors.initials: M
authors.lastname: Biel
authors.forename: Franz
authors.initials: F
authors.lastname: Hofmann
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D015221:Potassium Channels; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D016875:tau Proteins / Q000737:chemistry
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D018076:DNA, Complementary; C578710:HCN2 protein, human; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D016875:tau Proteins; D007532:Isoleucine; D010649:Phenylalanine; D000242:Cyclic AMP; D007930:Leucine