The Journal of General Physiology
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Published 1 February 2001. doi:10.1085/jgp.117.2.91
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© The Rockefeller University Press, 0022-1295/2001//91/ $5.00
Journal of General Physiology, Volume 117, Number 2, 2001

Original Article

Blocker State Dependence and Trapping in Hyperpolarization-Activated Cation Channels

Evidence for an Intracellular Activation Gate



Ki Soon Shina, Brad S. Rothberga, and Gary Yellena

a Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115

Hyperpolarization-activated cation currents (Ih) are key determinants of repetitive electrical activity in heart and nerve cells. The bradycardic agent ZD7288 is a selective blocker of these currents. We studied the mechanism for ZD7288 blockade of cloned Ih channels in excised inside-out patches. ZD7288 blockade of the mammalian mHCN1 channel appeared to require opening of the channel, but strong hyperpolarization disfavored blockade. The steepness of this voltage-dependent effect (an apparent valence of ~4) makes it unlikely to arise solely from a direct effect of voltage on blocker binding. Instead, it probably indicates a differential affinity of the blocker for different channel conformations. Similar properties were seen for ZD7288 blockade of the sea urchin homologue of Ih channels (SPIH), but some of the blockade was irreversible. To explore the molecular basis for the difference in reversibility, we constructed chimeric channels from mHCN1 and SPIH and localized the structural determinant for the reversibility to three residues in the S6 region likely to line the pore. Using a triple point mutant in S6, we also revealed the trapping of ZD7288 by the closing of the channel. Overall, the observations led us to hypothesize that the residues responsible for ZD7288 block of Ih channels are located in the pore lining, and are guarded by an intracellular activation gate of the channel.

Key Words: mHCN1 • SPIH • ZD7288 • pore

© 2001 The Rockefeller University Press


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J. Wang, S. Chen, and S. A. Siegelbaum
Regulation of Hyperpolarization-Activated Hcn Channel Gating and Camp Modulation Due to Interactions of Cooh Terminus and Core Transmembrane Regions
J. Gen. Physiol., September 1, 2001; 118(3): 237 - 250.
[Abstract] [Full Text] [PDF]


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