Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K+ channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca2+ conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca2+-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283–A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.
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1 July 2002
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June 24 2002
Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel
Manuel Simoes,
Manuel Simoes
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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Line Garneau,
Line Garneau
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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Hélène Klein,
Hélène Klein
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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Umberto Banderali,
Umberto Banderali
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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Fadi Hobeila,
Fadi Hobeila
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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Benoit Roux,
Benoit Roux
2Weill Medical College of Cornell University, New York, NY 10021
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Lucie Parent,
Lucie Parent
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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Rémy Sauvé
Rémy Sauvé
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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Manuel Simoes
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
Line Garneau
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
Hélène Klein
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
Umberto Banderali
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
Fadi Hobeila
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
Benoit Roux
2Weill Medical College of Cornell University, New York, NY 10021
Lucie Parent
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
Rémy Sauvé
1Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7
Address correspondence to Dr. Rémy Sauvé, Département de Physiologie, Université de Montréal C.P. 6128, Succursale Centre-ville Montréal, Quebec, Canada H3C 3J7. Fax: (514) 343-7146; E-mail: [email protected]
*
Abbreviations used in this paper: EDHF, endothelium-derived hyperpolarizing factor; EPR, electron paramagnetic spectroscopy; MTS, methanethiosulfonate, MTSES, sodium (2-sulfonatoethyl) methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide; SCAM, scanning mutagenesis.
Received:
March 04 2002
Revision Received:
May 22 2002
Accepted:
May 29 2002
Online ISSN: 1540-7748
Print ISSN: 0022-1295
The Rockefeller University Press
2002
J Gen Physiol (2002) 120 (1): 99–116.
Article history
Received:
March 04 2002
Revision Received:
May 22 2002
Accepted:
May 29 2002
Citation
Manuel Simoes, Line Garneau, Hélène Klein, Umberto Banderali, Fadi Hobeila, Benoit Roux, Lucie Parent, Rémy Sauvé; Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel . J Gen Physiol 1 July 2002; 120 (1): 99–116. doi: https://doi.org/10.1085/jgp.20028586
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