Among ion channels involved in the control of neuronal activity, small conductance calcium-activated potassium channels (SK) represent an interesting therapeutic target. Indeed, they underlie medium duration afterhyperpolarizations (mAHPs) in many types of neurons, thus inhibiting cell excitability. Three subtypes of SK subunits, SK1, SK2 and SK3, have been cloned and are expressed differentially within the central nervous system (CNS). Blocking SK channels might be beneficial in the treatment of several CNS disorders such as depression (SK3), Parkinson's disease (SK3) and cognitive disorders (SK2). So far, the prototypical blocker of SK channels is apamin, an octadecapeptide from bee venom. We have recently shown that apamin blocks SK channels by binding to a site distinct from that used by classical pore blockers such as tetraethylammonium (TEA) (Lamy et al. J. Biol. Chem. 2010, 285, 27067-77). We have also demonstrated that the nonpeptide blocker N-methyl-laudanosine (NML) (Scuvée-Moreau et al. J. Pharmacol. Exp. Ther. 2002, 302, 1176-83) competes for the binding site of the toxin. Further, our research team has recently shown that the sigma agonist 1,3-di-o-tolyl-guanidine (DTG) directly blocks SK currents in a voltage-independent manner (Lamy et al. Eur. J. Pharmacol. 2010, 641, 23-8). We have combined patch clamp experiments on cell lines with molecular modelling and mutagenesis, to try to identify the site where DTG blocks. DTG was found to be equipotent on wild-type (WT) and apamin-insensitive (e.g. SK2H337N) channels. Moreover, mutated channels with increased sensitivity to TEA (SK3V520F: mean IC₅₀ of TEA: 0.34 mM versus 11 mM for WT channels) were blocked by DTG with the same potency as WT channels. Thus, DTG does not seem to share the site of either apamin or TEA. Modelling data were in agreement with this possibility because of the identification of various potential binding sites. Although preliminary, these results suggest the existence of yet another binding site in the outer pore region of SK channels.