Submitted on August 1, 2007
Accepted on October 26, 2007
Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions
Ka-Wing Au1, Chung-Wah Siu2, Chu-Pak Lau3, Hung-Fat Tse3, and Ronald A Li4*
1 Medicine, The University of Hong Kong, Hong Kong; Institute of Cardiovascular Science and Medicine, University of Hong Kong, Hong Kong
2 Medicine, The University of Hong Kong, Hong Kong; Cell Biology and Human Anatomy, University of California, Davis, United States; Stem Cell Program, University of California, Davis, California, United States
3 Medicine, The University of Hong Kong, Hong Kong
4 Medicine, The University of Hong Kong, Hong Kong; Cell Biology and Human Anatomy, University of California, Davis, United States; Stem Cell Program, University of California, Davis, California, United States; Pediatric Regenerative Medicine, Shriners Hospital for Children of North America, Sacramento, California, United States
* To whom correspondence should be addressed. E-mail: ronli{at}ucdavis.edu.
The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane current If that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence the 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 to 345) of HCN1-R ("R" for resistance to sulfhydryl-reactive agents) channels, whose 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 the divalent Cd2+(I100µM Cd/IControl, -140mV= 67.6 ± 2.9%, 109.3 ± 3.1% and 103.8 ± 1.7%, respectively). Externally-applied MTSEA, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing I-140mV (to 43.7 ± 6.5%), causing a hyperpolarizing steady-state activation shift (
V1/2~6mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that the pore residues 339 to 345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.
