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

被引:8
作者
Au, Ka-Wing [1 ,2 ]
Siu, Chung-Wah [1 ,3 ,4 ]
Lau, Chu-Pak [1 ]
Tse, Hung-Fat [1 ]
Li, Ronald A. [1 ,3 ,4 ,5 ]
机构
[1] Univ Hong Kong, Dept Med, Queen Mary Hosp, Hong Kong, Hong Kong, Peoples R China
[2] Univ Hong Kong, Inst Cardiovasc Sci & Med, Hong Kong, Hong Kong, Peoples R China
[3] Univ Calif Davis, Stem Cell Program, Davis, CA 95616 USA
[4] Univ Calif Davis, Dept Cell Biol & Human Anat, Davis, CA 95616 USA
[5] Shriners Hosp Children N Amer, Inst Pediat Regenerat Med, Sacramento, CA USA
来源
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY | 2008年 / 294卷 / 01期
关键词
hyperpolarization-activated cyclic nucleotide; cysteine mutagenesis; sulfhydryl modification;
D O I
10.1152/ajpcell.00340.2007
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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 mu MCd2+/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: similar to 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.
引用
收藏
页码:C136 / C144
页数:9
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