Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

被引:39
作者
Dodier, Y [1 ]
Banderali, U [1 ]
Klein, H [1 ]
Topalak, Ö [1 ]
Dafi, O [1 ]
Simoes, M [1 ]
Bernatchez, G [1 ]
Sauvé, R [1 ]
Parent, L [1 ]
机构
[1] Univ Montreal, Fac Med, Dept Physiol, Membrane Prot Study Grp GEPROM, Montreal, PQ H3C 3J7, Canada
关键词
D O I
10.1074/jbc.M310534200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (Glu(515)) to the beginning of S6 (Ala(560)). 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 Pro(527) and Ile(541) was compatible with the presence of a alpha-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 Asp(542) (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 (Glu(515) to Tyr(526)) connected to a small helical segment of 15 amino acids ((527)PTALFSTFELFLT(539)) followed by two distinct coiled structures Ile(540)-Pro(544) (selectivity filter) and Ala(545) -Ile(557) before the beginning of S6.
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页码:6853 / 6862
页数:10
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