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J. Biol. Chem., Vol. 284, Issue 1, 389-403, January 2, 2009

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Hydrophobic Interactions as Key Determinants to the KCa3.1 Channel Closed Configuration

AN ANALYSIS OF KCa3.1 MUTANTS CONSTITUTIVELY ACTIVE IN ZERO Ca^2+*

From the Département de Physiologie, Groupe d'É_tude sur les Protéines Membranaires, Faculté de Médecine, Université de Montréal, Montréal, Québec H3C 3J7, Canada

In this study we present evidence that residue Val²⁸² in the S6 transmembrane segment of the calcium-activated KCa3.1 channel constitutes a key determinant of channel gating. A Gly scan of the S6 transmembrane segment first revealed that the substitutions A279G and V282G cause the channel to become constitutively active in zero Ca²⁺. Constitutive activity was not observed when residues extending from Cys²⁷⁶ to Ala²⁸⁶, other than Ala²⁷⁹ and Val²⁸², were substituted to Gly. The accessibility of Cys engineered at Val²⁷⁵ deep in the channel cavity was next investigated for the ion-conducting V275C/V282G mutant and closed V275C channel in zero Ca²⁺ using Ag⁺ as probe. These experiments demonstrated that internal Ag⁺ ions have free access to the channel cavity independently of the channel conducting state, arguing against an activation gate located at the S6 segment C-terminal end. Experiments were also conducted where Val²⁸² was substituted by residues differing in size and/or hydrophobicity. We found a strong correlation between constitutive activity in zero Ca²⁺ and the hydrophobic energy for side chain burial. Single channel recordings showed finally that constitutive activation in zero Ca²⁺ is better explained by a model where the channel is locked in a low conducting state with a high open probability rather than resulting from a change in the open/closed energy balance that would favor channel openings to a full conducting state in the absence of Ca²⁺. We conclude that hydrophobic interactions involving Val²⁸² constitute key determinants to KCa3.1 gating by modulating the ion conducting state of the selectivity filter through an effect on the S6 transmembrane segment.

Received for publication, July 24, 2008 , and in revised form, October 24, 2008.

^* This work was supported by grants from the Canadian Institutes of Health Research (MOP 7769), from the Canadian Cystic Fibrosis Foundation and from the Canadian Heart & Stroke Foundation. 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 To whom correspondence should be addressed: Groupe d'É_tude sur les Protéines Membranaires, Département de Physiologie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada. E-mail: remy.sauve{at}umontreal.ca.

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