Analysis of the blocking activity of charybdotoxin homologs and iodinated derivatives against Ca2+-activated K+ channels.
Two charybdotoxin peptides were purified from venom of the Israeli scorpion, Leiurus quinquestriatus hebraeus. Microsequencing of the most abundant toxin, ChTX-Lq1, revealed identity with the 37-residue peptide previously sequenced by Gimenez-Gallego et al. [Gimenez-Gallego, G., et al., Proc. Natl. Acad. Sci. USA 85:3329-3333 (1988)]. Sequence data on the minor peptide, ChTX-Lq2, showed substantial homology to ChTX-Lq1 with differences observed at eight positions. These two charybdotoxin sequences, along with that of noxiustoxin, define a distinct family of scorpion peptide toxins with activity against K+ channels. Both charybdotoxin homologs inhibited Ca2+-dependent K+ efflux from human erythrocytes with similar potency, K0.5 approximately 40 nM. In planar bilayer assays of single K(Ca) channels from rat muscle, ChTX-Lq1 and ChTX-Lq2 blocked with intrinsic Kd's of 1.3 and 43 nM, respectively, in the presence of 50 mM external KCl. A new application of dwell-time histogram analysis of single-channel blocking events was used to characterize the kinetic homogeneity of toxin samples and the blocking kinetics of ChTX derivatives. The lower blocking affinity of ChTX-Lq2 was the combined result of a faster dissociation rate and a slower association rate as compared to ChTX-Lq1. The blocking activity of two mono-iodinated derivatives of ChTX-Lq1 was also analyzed. Blocked dwell-time histograms of the iodinated peptides were characterized by predominately brief (0.2-2 sec) blocking events in comparison to the native toxin (20 sec). Histogram analysis revealed that mono-iodination of ChTX-Lq1 impairs blocking activity by adverse effects on both dissociation and association rate constants. Frequency density histograms of single channel blocking events provide a sensitive assay of toxin purity suitable for quantitating structure-activity relationships of charybdotoxin derivatives.
Lucchesi
K
K
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Ravindran
A
A
Young
H
H
Moczydlowski
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D018999:Charybdotoxin; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D012604:Scorpion Venoms / Q000378:metabolism / Q000494:pharmacology*; D012605:Scorpions
D007457:Iodine Radioisotopes; C106614:Lq2 protein, Leiurus quinquestriatus; D015221:Potassium Channels; D012604:Scorpion Venoms; D018999:Charybdotoxin; D011188:Potassium; D002118:Calcium