TY - JOUR T1 - Negative Gating Modulation by (R)-N-(Benzimidazol-2-yl)-tetrahydro-1-naphtylamine (NS8593) Depends on Residues in the Inner Pore Vestibule: Pharmacological Evidence of Deep-Pore Gating of KCa2 Channels JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.110.069807 SP - mol.110.069807 AU - David Paul Jenkins AU - Dorte Strobaek AU - Charlotte Hougaard AU - Marianne L. Jensen AU - Rene Hummel AU - Ulrik S. Sorensen AU - Palle Christophersen AU - Heike Wulff Y1 - 2011/01/01 UR - http://molpharm.aspetjournals.org/content/early/2011/03/01/mol.110.069807.abstract N2 - Acting as a negative gating modulator, NS8593 shifts the apparent Ca2+-dependence of the small-conductance Ca2+-activated K+ channels KCa2.1-2.3 to higher Ca2+ concentrations. Similar to the positive KCa channel gating-modulators 1-EBIO and CyPPA the binding site for NS8593 has been assumed to be located in the C-terminal region, where these channels interact with their calcium sensor calmodulin. However, by employing a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the KCa2 pore. For example, when we transferred the C-terminus from the NS8593-insensitive intermediate-conductance 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 to KCa3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis we subsequently identified two specific residues in the inner vestibule of KCa2.3 (S507 and A532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in KCa3.1 (T250 and V275) made KCa2.3 insensitive to NS8593, while introduction of S and A into KCa3.1 was sufficient to render this channel highly sensitive to NS8593. Interestingly, the same two residue positions have previously been 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 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. ER -