Direct block of native and cloned (Kir2.1) inward rectifier K+ channels bychloroethylclonidine

1 We have investigated the inhibition of inwardly rectifying potassium chan nels by the alpha-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flex or digitorum brevis muscle and whole-cell patch-clamp of cell lines transfe cted with Kir2.1 (IRK1). 2 In skeletal muscle and at a membrane potential of -50 mV, chloroethylclon idine (CEC), an agonist at alpha(2)-adrenergic receptors and an antagonist at alpha(1x)-receptors, was found to inhibit the inward rectifier current w ith a Ki of 30 mu M. 3 The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive t o high concentrations of alpha(1)-(prazosin) or alpha(2)-(rauwolscine) anta gonists. 4 The degree of current inhibition by CEC was found to vary with the membra ne potential (approximately 70% block at -50 mV c.f. similar to 10% block a t -190 mV). The kinetics of this voltage dependence were further investigat ed using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be similar to 8 s at 0 mV, and the rate of unblock was descri bed by the relationship tau = exp((Vm + 149)/22) s. 5 This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding s ite (aspartate 172) was mutated to asparagine. 6 The data suggest that the clonidine-like imidazoline compound, CEC, inhib its inward rectifier K+ channels independently of alpha-receptors by direct ly blocking the channel pore, possibly at an intracellular polyamine bindin g site.