title: An arginine residue in the pore region is a key determinant of chloride dependence in cardiac pacemaker channels.
abstract: The modulation of ion channel activity by extracellular ions plays a central role in the control of heart function. Here, we show that the sinoatrial pacemaker current I(f) is strongly affected by the extracellular Cl- concentration. We investigated the molecular basis of the Cl- dependence in heterologously expressed hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that represent the molecular correlate of I(f). Currents carried by the two cardiac HCN channel isoforms (HCN2 and HCN4) showed the same strong Cl- dependence as the sinoatrial I(f) and decreased to about 10% in the absence of external Cl-. In contrast, the neuronal HCN1 current was reduced to only 50% under the same conditions. Depletion of Cl- did not affect the voltage dependence of activation or the ion selectivity of the channels, indicating that the reduction of I(f) was caused by a decrease of channel conductance. A series of chimeras between HCN1 and HCN2 was constructed to identify the structural determinants underlying the different Cl- dependence of HCN1 and HCN2. Exchange of the ion-conducting pore region was sufficient to switch the Cl- dependence from HCN1- to HCN2-type and vice versa. Replacement of a single alanine residue in the pore of HCN1 (Ala-352) by an arginine residue present in HCN2 at equivalent position (Arg-405) induced HCN2-type chloride sensitivity in HCN1. Our data indicate that Arg-405 is a key component of a domain that allosterically couples Cl- binding with channel activation.
authors.affiliation: Department Pharmazie - Pharmakologie für Naturwissenschaften, Ludwig-Maximilians-Universität München, Butenandtstr. 7-13, 81377 München, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Wahl-Schott
authors.forename: Ludwig
authors.initials: L
authors.lastname: Baumann
authors.forename: Xiangang
authors.initials: X
authors.lastname: Zong
authors.forename: Martin
authors.initials: M
authors.lastname: Biel
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels; D005260:Female; D006321:Heart / Q000502:physiology*; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012849:Sinoatrial Node / Q000166:cytology / Q000378:metabolism*
chemical_list: D002712:Chlorides; D054815:Cyclic Nucleotide-Gated Cation Channels; C578665:HCN1 protein, human; C578710:HCN2 protein, human; C578666:Hcn1 protein, mouse; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D001120:Arginine