PEPTIDE PROBE OF RYANODINE RECEPTOR FUNCTION - IMPERATOXIN-A, A PEPTIDE FROM THE VENOM OF THE SCORPION PANDINUS-IMPERATOR, SELECTIVELY ACTIVATES SKELETAL-TYPE RYANODINE RECEPTOR ISOFORMS

We have used [H-3]ryanodine binding experiments and single channel recordings to provide convergent descriptions of the effect of imperatoxin A (IpTx(a)), a similar to 5-kDa peptide from the venom of the scorpion Pandinus imperator (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Ntl, Acad. Sc. U.S.A. 89, 12185-12189) on Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). At nanomolar concentrations, IpTx(a) increased the binding of [H-3]ryanodine to skeletal SR and, to a lesser extent, to cerebellum microsomes. The activating effect of IpTx(a) on skeletal SR was Ca2+-dependent, synergized by caffeine, and independent of other modulators of RyRs. However, IpTx(a) had negligible effects on tissues where the expression of skeletal-type RyR isoforms (RyR1) is small or altogether absent, i.e. cardiac, cerebrum, and liver microsomes. Thus, IpTx(a) may be used as a ligand capable of discriminating between RyR isoforms with nanomolar affinity, IpTx(a) increased the open probability (P-o) of rabbit skeletal muscle RyRs by increasing the frequency of open events and decreasing the duration of the closed lifetimes. This activating effect was dose-dependent (ED(50) = 10 mM), had a fast onset, and was fully reversible, Purified RyR from solubilized skeletal SR displayed high affinity for [H-3]ryanodine with a K-D of 6.1 nM and B-max of similar to 30 pmol/mg of protein. IpTx(a) increased [H-3]ryanodine binding noncompetitively by increasing B-max to similar to 60 pmol/mg of protein. These results suggested the presence of an IpX(a)-binding site on the RyR or a closely associated regulatory protein, This site appears to be distinct from the caffeine- and adenine nucleotide-regulatory sites, IpTx(a) may prove a useful tool to identify regulatory domains critical for channel gating and to dissect the contribution of skeletal-type RyRs to intracellular Ca2+ waveforms generated by stimulation of different RyR isoforms.