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J. Biol. Chem., Vol. 280, Issue 25, 24064-24071, June 24, 2005

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Gating Deficiency in a Familial Hemiplegic Migraine Type 1 Mutant P/Q-type Calcium Channel^*

Curtis F. Barrett, Yu-Qing Cao, and Richard W. Tsien

From the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305

Familial hemiplegic migraine type 1 (FHM1) arises from missense mutations in the gene encoding _1A, the pore-forming subunit of P/Q-type calcium channels. The nature of the channel disorder is fundamental to the disease, yet is not well understood. We studied how the most prevalent FHM1 mutation, a threonine to methionine substitution at position 666 (TM), affects both ionic current and gating current associated with channel activation, a previously unexplored feature of P/Q channels. Whole-cell currents were measured in HEK293 cells expressing channels containing either wild-type (WT) or TM _1A. Calcium currents were significantly smaller in cells expressing TM channels, consistent with previous reports. In contrast, surface expression of TM channels, measured by immunostaining against an extracellular epitope, was not decreased, and Western blots demonstrated that TM _1A subunits were expressed as full-length proteins. WT and TM gating currents were isolated by replacing Ca²⁺ with the nonpermeant cation La³⁺. The gating currents generated by the mutant channels were one-third that of WT, a deficiency sufficient to account for the observed attenuation in calcium current; the remaining gating current was no different in kinetics or voltage dependence. Thus, the decreased calcium influx seen with TM channels can be attributed to a reduced number of channels available to undergo the voltage-dependent conformational changes needed for channel opening, not to fewer channel proteins expressed on the cell surface. This identification of an intrinsic defect in FHM1 mutant channels helps explain their impact on neurotransmission when they occupy type-specific slots for P/Q channels at central nerve terminals.

Received for publication, February 28, 2005 , and in revised form, March 24, 2005.

^* This work was supported by National Institutes of Health Grant NS24067 (to R. W. T.), the George D. Smith Professorship (to R. W. T.), a Stanford Dean's Fellowship and an individual National Research Service Award (to Y.-Q. C.), and a NHLBI National Institutes of Health training grant (to C. F. B.). 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.

The on-line version of this article (available at http://www.jbc.org/) contains supplemental Figs. S1 and S2.

To whom correspondence should be addressed: Stanford University School of Medicine, Beckman Center, Rm. B105, Stanford, CA 94305-5345. Tel.: 650-725-7557; Fax: 650-725-8021; E-mail: rwtsien{at}stanford.edu.

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