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J. Biol. Chem., Vol. 279, Issue 8, 6853-6862, February 20, 2004

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Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis^*

Yolaine Dodier, Umberto Banderali, Hélène Klein, Özlem Topalak, Omar Dafi, Manuel Simoes, Gérald Bernatchez, Rémy Sauvé, and Lucie Parent, A senior scholar from the Fonds de la Recherche en Santé du Québec

From the Department of Physiology, Membrane Protein Study Group (GEPROM), Faculty of Medicine, Université de Montréal, Montréal, Québec H3C 3J7, Canada

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu⁵¹⁵) to the beginning of S6 (Ala⁵⁶⁰). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro⁵²⁷ and Ile⁵⁴¹ was compatible with the presence of a -helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp⁵⁴² (high Ca²⁺ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu⁵¹⁵ to Tyr⁵²⁶) connected to a small helical segment of 15 amino acids (⁵²⁷PTALFSTFELFLT⁵³⁹) followed by two distinct coiled structures Ile⁵⁴⁰-Pro⁵⁴⁴ (selectivity filter) and Ala⁵⁴⁵-Ile⁵⁵⁷ before the beginning of S6.

Received for publication, September 23, 2003 , and in revised form, November 18, 2003.

^* This work was supported by grants from the Kidney Foundation of Canada and the Canadian Institutes of Health Research (MOP 13390 and MMA 62995). 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.

To whom correspondence should be addressed. Tel.: 514-343-6673; Fax: 514-343-7146; E-mail: lucie.parent{at}umontreal.ca

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