{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T10:33:19Z","timestamp":1761561199611},"reference-count":32,"publisher":"American Physiological Society","issue":"1","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Cell Physiology"],"published-print":{"date-parts":[[2008,1]]},"abstract":"<jats:p> 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\u2013345) 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<jats:sup>2+<\/jats:sup> (current with 100 \u03bcM Cd<jats:sup>2+<\/jats:sup>\/control current at \u2212140 mV = 67.6 \u00b1 2.9%, 109.3 \u00b1 3.1%, and 103.8 \u00b1 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at \u2212140 mV (to 43.7 \u00b1 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: \u223c6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339\u2013345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible. <\/jats:p>","DOI":"10.1152\/ajpcell.00340.2007","type":"journal-article","created":{"date-parts":[[2007,11,8]],"date-time":"2007-11-08T02:05:49Z","timestamp":1194487549000},"page":"C136-C144","source":"Crossref","is-referenced-by-count":8,"title":["Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions"],"prefix":"10.1152","volume":"294","author":[{"given":"Ka-Wing","family":"Au","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chung-Wah","family":"Siu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chu-Pak","family":"Lau","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hung-Fat","family":"Tse","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ronald A.","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"24","reference":[{"key":"R1","doi-asserted-by":"crossref","unstructured":"Azene EM, Sang D, Tsang SY, Li RA. 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