Di-substituted cyclohexyl derivatives bind to two identical sites with positive cooperativity on the voltage-gated...

Di-substituted cyclohexyl (DSC) derivatives inhibit the voltage-gated potassium channel, K(v)1.3, and have immunosuppressant activity (Schmalhofer et al. (2002) Biochemistry 41, 7781-7794). This class of inhibitors displays Hill coefficients of near 2 in functional assays, and trans DSC analogues appear to selectively interact with K(v)1.3 channel conformations related to C-type inactivation. To further understand the details of the DSC inhibitor interaction with potassium channels, trans-1-(N-n-propylcarbamoyloxy)-4-phenyl-4-(3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl)cyclo-hexane (trans-NPCO-DSC) was radiolabeled with tritium, and its binding characteristics to K(v)1.3 channels were determined. Specific binding of [(3)H]-trans-NPCO-DSC to K(v)1.3 channels is a saturable, time-dependent, and fully reversible process. Saturation binding isotherms and competition binding experiments are consistent with the presence of two receptor sites for DSC derivatives on the K(v)1.3 channel that display positive allosteric cooperativity. The high affinity interaction of [(3)H]-trans-NPCO-DSC with K(v)1.3 channels appears to correlate with the rates of C-type inactivation of the channel. These data, taken together, mark the first demonstration of the existence of multiple binding sites for an inhibitor of an ion channel and suggest that the high affinity interaction of trans-NPCO-DSC and similar inhibitors with K(v)1.3 channels could be exploited for the development of selective molecules that target this protein.