title: Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions.
abstract: Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339-345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd(2+) (current with 100 microM Cd(2+)/control current at -140 mV = 67.6 +/- 2.9%, 109.3 +/- 3.1%, and 103.8 +/- 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at -140 mV (to 43.7 +/- 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: approximately 6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339-345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.
authors.affiliation: Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong.
authors.forename: Ka-Wing
authors.initials: KW
authors.lastname: Au
authors.forename: Chung-Wah
authors.initials: CW
authors.lastname: Siu
authors.forename: Chu-Pak
authors.initials: CP
authors.lastname: Lau
authors.forename: Hung-Fat
authors.initials: HF
authors.lastname: Tse
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019256:Cadmium Chloride / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D008845:Microinjections; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes; D010539:Permeability; D015221:Potassium Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019163:Reducing Agents / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015221:Potassium Channels; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D019256:Cadmium Chloride; D003545:Cysteine; D004229:Dithiothreitol