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

We have investigated the inhibition of inwardly rectifying potassium channels by the α‐adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two‐electrode voltage‐clamp of rat isolated flexor digitorum brevis muscle and whole‐cell patch‐clamp of cell lines transfected with Kir2.1 (IRK1). In skeletal muscle and at a membrane potential of −50 mV, chloroethylclonidine (CEC), an agonist at α 2 ‐adrenergic receptors and an antagonist at α 1x ‐receptors, was found to inhibit the inward rectifier current with a K i of 30 μ M . The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of α 1 ‐(prazosin) or α 2 ‐(rauwolscine) antagonists. The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at −50 mV c.f . ∼10% block at −190 mV). The kinetics of this voltage dependence were further investigated 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 ∼8 s at 0 mV, and the rate of unblock was described by the relationship τ=exp(( Vm +149)/22) s. 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 site (aspartate 172) was mutated to asparagine. The data suggest that the clonidine‐like imidazoline compound, CEC, inhibits inward rectifier K + channels independently of α‐receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.British Journal of Pharmacology (1999) 128 , 760–766; doi: 10.1038/sj.bjp.0702819