A point mutation in the maxi-K clone dSlo forms a high affinity site for charybdotoxin.
This work investigated the interaction of CTX with two cloned analogues of the maxi-K channel, dSlo and hSlo. dSlo has been reported to be CTX insensitive. Single channel analysis revealed that dSlo was weakly blocked by the toxin, with a very high K(D) of 5.8 microM. The hSlo channels bound wild-type, recombinant CTX with high affinity and in a bimolecular fashion, and displayed a half-blocking concentration (K(D)) of 36 nM. A glutamate residue was substituted for the wild-type threonine at position 290 in dSlo. The mutant channel was expressed in COS-7 cells and reconstituted into lipid bilayers for single channel analysis. The mutant channel bound wild-type, recombinant CTX with high affinity and in a bimolecular fashion, and displayed a half-blocking concentration (K(D)) of 23 nM. Changing just one residue from threonine to glutamate at position 290 in dSlo changed the affinity of the channel's CTX-receptor over 100-fold. Kinetic analysis revealed that this large increase in affinity was due to decreasing the dissociation rate of the toxin. These results suggest that a CTX receptor does exist in the dSlo channel mouth and that the threonine at position 290 destabilizes the toxin on the binding site.
Myers
M P
MP
Department of Pharmacology aond Physiology, University of Rochester School of Medicine and Dentistry, NY 14642-8711, USA. adric@ucla.edu
Stampe
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D018999:Charybdotoxin / Q000493:pharmacokinetics / Q000494:pharmacology*; D003001:Cloning, Molecular; D004594:Electrophysiology / Q000379:methods; D018698:Glutamic Acid; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D024681:Potassium Channels, Calcium-Activated; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013912:Threonine; D014162:Transfection
D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D011994:Recombinant Proteins; D018999:Charybdotoxin; D013912:Threonine; D018698:Glutamic Acid