The mechanism of inhibition of ryanodine receptor channels by imperatoxin I, a heterodimeric protein from the scorpion Pandinus imperator.

We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an approximately 15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta-bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta-bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.

Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism.

1. The regulation of the cardiac Ca2+ release channel-ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1-20 U ml-1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)-dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/- 0.85 and 3.9 +/- 0.65 nM; Bmax (the maximal receptor density), 1.45 +/- 0.14 and 1.47 +/- 0.12 pmol mg-1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were 'dormant' due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non-hydrolysable ATP analogue adenylyl[beta, gamma-methylene]-diphosphate (AMP-PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation-dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.

Inhibition of dihydropyridine-sensitive calcium channels by the plant alkaloid ryanodine.

At micromolar concentrations, ryanodine interacts with the dihydropyridine receptor of rabbit skeletal muscle transverse tubules. Ryanodine displaces specifically bound [3H]PN200-110 with an apparent inhibition constant of approx. 95 microM and inhibits dihydropyridine-sensitive calcium channels in the same preparation with an IC50 of approx. 45 microM. These concentrations of ryanodine are approximately three orders of magnitude higher than those required to saturate binding of the alkaloid to the ryanodine receptor of sarcoplasmic reticulum and to open the calcium release channel of sarcoplasmic reticulum (i.e. 20 nM (1988) J. Gen. Physiol. 92, 1-26). Thus at sufficiently high dose, ryanodine may affect SR as well as plasma membrane Ca permeabilities.