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J. Biol. Chem., Vol. 280, Issue 18, 18253-18264, May 6, 2005

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Molecular Compatibility of the Channel Gate and the N Terminus of S5 Segment for Voltage-gated Channel Activity^*

Marco Caprini¶, Marianna Fava, Pierluigi Valente, Gregorio Fernandez-Ballester, Carmela Rapisarda, Stefano Ferroni, and Antonio Ferrer-Montiel||

From the Department of Human and General Physiology, University of Bologna, Via San Donato 19/2, 40127 Bologna, Italy and the Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Alicante, Spain

Voltage-gated ion channels are modular proteins designed by the structural linkage of a voltage sensor and a pore domain. The functional coupling of these two protein modules is a subject of intense research. A major focus has been directed to decipher the role of the S4-S5 linker and the C-end of the inner pore helix in channel gating. However, the contribution of the cytosolic N terminus of S5 remains elusive. To address this issue, we used a chimeric subunit that linked the voltage sensor of the Shaker channel to the prokaryotic KcsA pore domain (denoted as Shaker-KcsA). This chimera preserved the Shaker sequences at both the N terminus of S5 and the C-end of S6. Chimeric Shaker-KcsA subunits did not form functional homomeric channels but were synthesized, folded, and trafficked to the cell surface, as evidenced by their co-assembly with Shaker wild type subunits. Sequential substitution of Shaker amino acids at the C-end of S6 and the N terminus of S5 by the corresponding KcsA created voltage-sensitive channels with voltage-dependent properties that asymptotically approached those of the wild type Shaker channel. Noteworthy, substitution of the region encompassing Phe⁴⁰¹-Phe⁴⁰⁴ at the N-end of Shaker S5 by KcsA residues resulted in a significant gain in voltage sensitivity of the chimeras. Furthermore, analysis of channel function at high [K⁺]_o revealed that the Phe⁴⁰¹-Phe⁴⁰⁴ region is an important molecular determinant for competent coupling of voltage sensing and pore opening. Taken together, these findings indicate that complete replacement of Shaker S5 and S6 by KcsA M1 and M2 is required for voltage-dependent gating of the prokaryotic channel. In addition, our results imply that the region encompassing Phe⁴⁰¹-Phe⁴⁰⁴ in Shaker is involved in protein-protein interactions with the voltage sensor, and signal to the Phe⁴⁰¹ in the S5 segment as a key molecular determinant to pair the voltage sensor and the pore domain.

Received for publication, November 29, 2004 , and in revised form, January 31, 2005.

^* This work was supported in part by Ministerio de Educacion Ciencia y Deporte Grant SAF 2003/0509, La Fundació La Caixa Grant 01/08-500, and Generalitat Valenciana Grant GV04B-0398 (to A. F. M.), and by a grant from Ministerio dell'Istruzione dell'Universita e della Ricerca (MIUR) (Italy) (to S. F. and C. R.). 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.

^¶ Supported by fellowships, "Assegni di Ricerca" and "Marco Polo," from the University of Bologna.

^|| To whom correspondence should be addressed: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Av. de la Universidad s/n, 03202 Elche (Alicante), Spain. Tel.: 34-96-665-8727; Fax: 34-96-665-8758; E-mail: aferrer{at}umh.es.

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