Pharmacological Evidence For Deep Pore Gating In SK Channels

Abstract

SK channels (KCa2.1-KCa2.3) are gated by Ca2+ through calmodulin bound to a domain (CaMBD) in the cytoplasmic C-terminus. The [Ca2+]i response curve is identical for all SK subtypes and a similar gating applies for the related IK channel (KCa3.1). Pharmacological intervention with SK/IK channels is achievable by peptides and synthetic pore blockers but also by modulators of the gating process. Positive modulators of IK and SK channels, such as 1-EBIO and NS309, induce a shift of the [Ca2+]i response curve towards lower [Ca2+]i and have been known for several years.Recently the “opposite” principle, negative gating modulation, was described for NS8593 (Strobaek et al., 2006). NS8593 selectively inhibits SK channels and in whole-cell patch-clamp experiments, using HEK293 cells transiently transfected with hSK3, a Kd value of 108±33 nM (n=12) was obtained, whereas hIK was insensitive to 10 μM NS8593. The positive modulation by 1-EBIO is mediated via the C-terminus (Pedarzani et al., 2005), but we have found that SK3 channels in which the C-terminus was substituted with the corresponding IK channel tail retained their NS8593-sensitivity (Biophys. J. 2008 94: 2183). Now we show, that amino acids in the deep pore are important for NS8593-induced inhibition: IK channels where T250, a residue just below the selectivity filter, or V275, in TM6, was substituted with the corresponding amino acids from SK3 became sensitive to NS8593 with Kd values of 513±274 nM (n=6) and 4181±1530 nM (n=9), respectively. Interestingly, these exact amino acids are essential for block of IK channels by TRAM-34 and clotrimazole (Wulff et al., 2001). Additional experiments will be conducted to determine whether NS8593 acts as a negative gating modifier on these IK mutants and whether these amino acids play a role in the normal gating process of SK channels.