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J. Biol. Chem., Vol. 279, Issue 16, 16832-16846, April 16, 2004

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Structural Elements of Instantaneous and Slow Gating in Hyperpolarization-activated Cyclic Nucleotide-gated Channels^*

Vincenzo Macri and Eric A. Accili

From the Ion Channel Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

Hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits produce a slowly activating current in response to hyperpolarization (I_f) and an instantaneous voltage-independent current (I_inst) when expressed in Chinese hamster ovary (CHO) cells. Here we found that a mutation in the S4-S5 linker of HCN2 (Y331D) produced an additional mixed cationic instantaneous current. However, this current was inhibited by external Cs⁺ like I_f and unlike I_inst. Together with a concomitant reduction in I_f, the data suggest that the Y331D mutation disrupted channel closing placing the channel in a "I_f -like," and not an "I_inst-like," state. The "I_f-like" instantaneous current represented 70% of total I_f over voltages ranging from +20 to -150 mV in high K⁺ solutions. I_f activated at more depolarized potentials and the activation curve was less steep, whereas deactivation was significantly slowed, consistent with the idea that the mutation inhibited channel closing. The data suggest that the mutation produced allosteric effects on the activation gate (S6 segment) and/or on voltage-sensing elements. We also found that decreases in the ratio of external K⁺/Na⁺ further disrupted channel closing in the mutant channel. Finally, our data suggest that the structures involved in producing I_inst are similar between the HCN1 and HCN2 isoforms and that excess HCN protein on the plasma membrane of CHO cells relative to native cells is not responsible for I_inst. The data are consistent with I_inst flowing through a "leaky" closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits.

Received for publication, January 16, 2004

^* This work was supported by grants from the Heart and Stroke Foundation of Canada, the Heart and Stroke Foundation of British Columbia and the Yukon, and the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 604-291-4574; Fax: 604-291-3040; E-mail: eaaccili{at}sfu.ca.

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