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J. Biol. Chem., Vol. 280, Issue 46, 38471-38477, November 18, 2005

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Structural Determinants of L-type Channel Activation in Segment IIS6 Revealed by a Retinal Disorder^*

Annette Hohaus, Stanislav Beyl1, Michaela Kudrnac1, Stanislav Berjukow, Eugen N. Timin, Rainer Marksteiner, Marion A. Maw¶2, and Steffen Hering3

From the Institute for Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, A-1090 Wien, InnovaCell Biotechnology GmbH, Mitterweg 24, A-6020 Innsbruck, and the ^¶Biochemistry Department, University of Otago, P.O. Box 56, Dunedin 901, Aotearoa, New Zealand

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the Ca_V1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the Ca_V1.2 channel. Substitution of Ile-781, the Ca_V1.2 residue corresponding to Ile-745 in Ca_V1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of Ca_V1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of Ca_V1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

Received for publication, June 28, 2005 , and in revised form, September 9, 2005.

^* This work was supported in part by a grant from Fonds zur Förderung der wissenschaftlichen Forschung (FWF) 15914 (to S. H.). 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.

¹ Both authors contributed equally to this work.

² Supported by the Health Research Council of New Zealand.

³ To whom correspondence should be addressed. Tel.: 43-14277-55310; Fax: 43-14277-9553; E-mail: steffen.hering{at}univie.ac.at.

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