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J. Biol. Chem., Vol. 283, Issue 13, 8136-8144, March 28, 2008

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Characterization of the Gating Brake in the I-II Loop of Ca_v3.2 T-type Ca²⁺ Channels^*

Imilla I. Arias-Olguín¹, Iuliia Vitko¹, Michal Fortuna, Joel P. Baumgart^¶, Svetlana Sokolova, Igor A. Shumilin^||, Amy Van Deusen, Manuel Soriano-García^**, Juan C. Gomora², and Edward Perez-Reyes^¶3

From the Departments of Pharmacology and ^||Molecular Physics and Biological Physics and the ^¶Neuroscience Graduate Program, University of Virginia, Charlottesville, Virginia 22908 and the Institutes of Cellular Physiology and ^**Chemistry, Universidad Nacional Autónoma de México, 04510 México D.F., México

Mutations in the I-II loop of Ca_v3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high -helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.

Received for publication, October 23, 2007 , and in revised form, January 16, 2008.

^* This work was supported by National Institutes of Health Grant NS038691 (to E. P.-R.) and Consejo Nacional de Ciencia y Tecnología Grant J50250-Q (to J. C. G.). 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 Fig. S1.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México D.F., México. Tel.: 5255-5622-5752; Fax: 5255-5622-5607; E-mail: jgomora{at}ifc.unam.mx. ³ To whom correspondence may be addressed: Dept. of Pharmacology, University of Virginia, Charlottesville, VA 22908. Tel.: 434-982-4440; Fax: 434-982-3878; E-mail: eperez{at}virginia.edu.

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