Negative Gating Modulation by (R)-N-(Benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) Depends on Residues in the Inner Pore Vestibule: Pharmacological Evidence of Deep-Pore Gating of KCa2 Channels

Abstract

Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca²⁺-dependence of the small-conductance Ca²⁺-activated K⁺ channels K_Ca2.1–2.3 to higher Ca²⁺ concentrations. Similar to the positive K_Ca channel-gating modulators 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca²⁺ sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the K_Ca2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance K_Ca3.1 channel to K_Ca2.3, the chimeric channel remained as sensitive to NS8593 as wild-type K_Ca2.3. In contrast, when we transferred the K_Ca2.3 pore to K_Ca3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of K_Ca2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in K_Ca3.1 (Thr250 and Val275) made K_Ca2.3 insensitive to NS8593, whereas introduction of serine and alanine into K_Ca3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of K_Ca3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the pore-loop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the K_Ca2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.