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Negative gating modulation by NS8593 depends on residues in the inner pore vestibule
Author(s) -
Jenkins David Paul,
Strøbæk Dorte,
Hougaard Charlotte,
Jensen Marianne,
Hummel Rene,
Sørensen Ulrik S.,
Christophersen Palle,
Wulff Heike
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1042.15
Subject(s) - gating , vestibule , modulation (music) , chemistry , biophysics , neuroscience , physics , biology , vestibular system , acoustics
Acting as a negative gating modulator, NS8593 shifts the apparent calcium‐dependence of the small conductance Ca 2+ ‐activated K + channels KCa2.1–2.3 to higher Ca 2+ concentrations. Similar to the positive gating‐modulators, EBIO and CyPPA, the binding site for NS8593 was assumed to be in the C‐terminus, where these channels interact with calmodulin. However, employing a progressive chimeric approach, we localized the site‐of‐action of NS8593 to the KCa2 pore. When we transferred the C‐terminus from the NS8593‐insensitive KCa3.1 channel to KCa2.3, the chimeric channel remained as sensitive to NS8593 as WT KCa2.3. In contrast, when we transferred the KCa2.3 pore, KCa3.1 became sensitive to NS8593. We subsequently identified two residues in the inner vestibule of KCa2.3 (S507 and A532) that mediated the effect of NS8593. Mutation of these residues to the corresponding residues in KCa3.1 (T250 and V275) made KCa2.3 insensitive, while introduction of S and A into KCa3.1 made it sensitive to NS8593. These same residues were previously found to mediate sensitivity of KCa3.1 to clotrimazole and TRAM‐34. The location of S507 in the pore‐loop, near the selectivity filter, and A532 in an adjacent position in the S6 segment, are within in the region predicted to contain the KCa2 channel gate. Based on these results, we propose that NS8593 mediated gating modulation occurs at a position deep within the inner pore vestibule.