SK channels are small conductance Ca²⁺-activated K⁺ channels important for the control of neuronal excitability and fine tuning of firing patterns and synaptic function (Stocker, 2004). The classical SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore blocking mechanism (Strong, 1990; Ishii et al. 1997). Recently, 1-EBIO (Pedarzani et al. 2001) and NS309 (Pedarzani et al. 2005) have been identified as SK channel enhancers or positive gating modulators, which work by increasing the apparent Ca²⁺ sensitivity of SK channels. Here we describe negative gating modulation as a novel principle for selective inhibition of SK channels. In whole-cell patch clamp experiments the benzimidazol-based compound NS8593 reversibly inhibited recombinant rat SK3-mediated currents with a K_i value of 90 ± 8 nM (n=33). However, in contrast to known pore blockers, NS8593 did not inhibit [¹²⁵I]apamin binding in assays performed on HEK293 cells expressing human (h) SK3 channels or rat striatal membranes. Using excised patches, it was demonstrated that NS8593 decreased the apparent Ca²⁺ sensitivity of hSK channels by shifting to the right the activation curve for Ca²⁺, without affecting the maximal Ca²⁺-activated SK current. In the absence of NS8593, hSK3 channels were highly sensitive to Ca²⁺ with an EC₅₀ of 0.43 µM and a Hill coefficient of 5.5 (n=17). In the presence of 3 µM NS8593, the channels had a reduced Ca²⁺ sensitivity with an EC₅₀ of 2 µM and a Hill coefficient of 2.8 (n=3). The shift in the Ca²⁺ activation curve indicated that the effect of NS8593 was pronounced at low Ca²⁺ concentrations, whereas the inhibition of hSK3 currents by NS8593 was fully abolished at 100 µM Ca²⁺. Similar Ca²⁺ activation curves were obtained for hSK1- and hSK2-mediated currents, showing that the affinity for NS8593 was Ca²⁺-dependent on these SK channel subtypes. The potency of NS8593 was similar at hSK1, hSK2, and hSK3 channels, but the compound did not affect hIK or hBK channels. The site of action of NS8593 was accessible from both sides of the membrane and the inhibition was prevented in the presence of high concentrations of the positive modulator NS309. NS8593 was further tested in whole-cell patch clamp experiments on mouse CA1 neurons in hippocampal slices. NS8593 strongly inhibited (84.7±1.4% inhibition at 3 µM, n=3; 86.7 ± 5.1% inhibition at 10 µM, n=6) the apamin- and tubocurarine-sensitive SK-mediated afterhyperpolarising current I_AHP elicited by 100 ms long depolarising pulses (as in Pedarzani et al. 2005).