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J. Biol. Chem., Vol. 279, Issue 5, 3635-3642, January 30, 2004

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The Predicted TM10 Transmembrane Sequence of the Cardiac Ca²⁺ Release Channel (Ryanodine Receptor) Is Crucial for Channel Activation and Gating^*

Ruiwu Wang, Jeff Bolstad, Huihui Kong, Lin Zhang, Cindy Brown, and S. R. Wayne Chen, Senior scholar of the Alberta Heritage Foundation for Medical Research.

From the Cardiovascular Research Group, Departments of Physiology and Biophysics, and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada

The predicted TM10 transmembrane sequence, ⁴⁸⁴⁴IIFDITFFFFVIVILLAIIQGLII⁴⁸⁶⁷, has been proposed to be the pore inner helix of the ryanodine receptor (RyR) and to play a crucial role in channel activation and gating, as with the inner helix of bacterial potassium channels. However, experimental evidence for the involvement of the TM10 sequence in RyR channel activation and gating is lacking. In the present study, we have systematically investigated the effects of mutations of each residue within the 24-amino acid TM10 sequence of the mouse cardiac ryanodine receptor (RyR2) on channel activation by caffeine and Ca²⁺. Intracellular Ca²⁺ release measurements in human embryonic kidney 293 cells expressing the RyR2 wild type and TM10 mutants revealed that several mutations in the TM10 sequence either abolished caffeine response or markedly reduced the sensitivity of the RyR2 channel to activation by caffeine. By assessing the Ca²⁺ dependence of [³H]ryanodine binding to RyR2 wild type and TM10 mutants we also found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca²⁺ and enhanced the basal activity of [³H]ryanodine binding. Furthermore, single I4862A mutant channels exhibited considerable channel openings and altered gating at very low concentrations of Ca²⁺. Our data indicate that the TM10 sequence constitutes an essential determinant for channel activation and gating, in keeping with the proposed role of TM10 as an inner helix of RyR. Our results also shed insight into the orientation of the TM10 helix within the RyR channel pore.

Received for publication, October 16, 2003 , and in revised form, October 28, 2003.

^* This work was supported by research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, Northwestern Territories, and Nunavut (to S. R. W. C.). 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: Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-4235; Fax: 403-283-4841; E-mail: swchen{at}ucalgary.ca.

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