Abstract

SK channels are small conductance Ca²⁺-activated K⁺ channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca²⁺ sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In wholecell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit ¹²⁵I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca²⁺ sensitivity by shifting the activation curve for Ca²⁺ to the right, only slightly affecting the maximal Ca²⁺-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca²⁺-dependently (K_d = 0.42, 0.60, and 0.73 μM, respectively, at 0.5 μM Ca²⁺), whereas the compound did not affect the Ca²⁺-activated K⁺ channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 μM.