A snake toxin inhibitor of inward rectifier potassium channel ROMK1

Mamba snake dendrotoxins have been used extensively in biochemical and physiological studies of K+ channels of the brain. Their known targets of inhibition have been Limited to the family of voltage-gated K+ channels. We report the isolation of a dendrotoxin inhibitor of ROMK1, a channel belonging to the inward rectifier family of K+ channels. The inhibitory activity, fractionated to purity with FPLC and HPLC, is identical to a previously identified delta-dendrotoxin. To verify that d-dendrotoxin blocks ROMK1 channels, a cDNA encoding the toxin was synthesized and recombinant toxin expressed in Escherichia coli. Electrophysiological recordings reveal that recombinant delta-dendrotoxin has a half-maximal inhibition constant (K-d) of 150 nM when applied to ROMK1 channels expressed in Xenopus laevis oocytes. That the delta-dendrotoxin binding site exists on separate K+ channel classes is shown by its high affinity for two of the voltage-gated family of channels, Kv1.1 (K-d < 0.1 nM) and Kv1.6 (K-d = 23 nM). Single amino acid substitutions in ROMK1 indicate that delta-dendrotoxin binds to the pore region of ROMK1 even though it does not completely block conduction through the pore. These results suggest that dendrotoxins inhibit K+ channels by recognizing the structurally conserved pore region of these channels.