Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis.
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 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 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.
Dodier
Yolaine
Y
Department of Physiology, Membrane Protein Study Group (GEPROM), Faculty of Medicine, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Banderali
Umberto
U
Klein
Hélène
H
Topalak
Ozlem
O
Dafi
Omar
O
Simoes
Manuel
M
Bernatchez
Gérald
G
Sauvé
Rémy
R
Parent
Lucie
L
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry; D015220:Calcium Channels / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D007477:Ions; D007532:Isoleucine / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D011392:Proline / Q000737:chemistry; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012984:Software; D050916:TRPV Cation Channels; D013997:Time Factors; D014982:Xenopus laevis
D000596:Amino Acids; D015220:Calcium Channels; D018076:DNA, Complementary; D007477:Ions; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; D007532:Isoleucine; D011392:Proline; D003545:Cysteine; D002118:Calcium