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This Article Full Text Full Text (PDF) All Versions of this Article: 294/1/C136    most recent 00340.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Au, K.-W. Articles by Li, R. A. Search for Related Content PubMed PubMed Citation Articles by Au, K.-W. Articles by Li, R. A.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

¹Department of Medicine, Queen Mary Hospital, and ²Institute of Cardiovascular Science and Medicine, University of Hong Kong, Hong Kong; ³Stem Cell Program and ⁴Department of Cell Biology and Human Anatomy, University of California, Davis; and ⁵Institute of Pediatric Regenerative Medicine, Shriners Hospital for Children of North America, Sacramento, California

Submitted 1 August 2007 ; accepted in final form 23 October 2007

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 Cd²⁺ (current with 100 µM Cd²⁺/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: 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.

hyperpolarization-activated cyclic nucleotide; cysteine mutagenesis; sulfhydryl modification