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

摘要

First published November?, 2007; doi:10.1152/ajpcell.00340.2007.-Hypeipolarization-ac-tivated 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 HCNl-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 HCNl-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd2+ (current with 100 muM 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 hyperpolar-izing 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.