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 delta-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 (Kd) 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 (Kd < 0.1 nM) and Kv1.6 (Kd = 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.
Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, New York 10021, USA.
J P
JP
Imredy
C
C
Chen
R
R
MacKinnon
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D004546:Elapid Venoms / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017815:Elapidae; D004594:Electrophysiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009865:Oocytes / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000494:pharmacology; D014982:Xenopus laevis
D004546:Elapid Venoms; D009498:Neurotoxins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; C020273:dendrotoxin