Multiple Actions of Imperatoxin A on Ryanodine Receptors

Abstract

Imperatoxin A is a high affinity activator of ryanodine receptors. The toxin contains a positively charged surface structure similar to that of the A fragment of skeletal dihydropyridine receptors (peptide A), suggesting that the toxin and peptide could bind to a common site on the ryanodine receptor. However, the question of a common binding site has not been resolved, and the concentration dependence of the actions of the toxin has not been fully explored. We characterize two novel high affinity actions of the toxin on the transient gating of cardiac and skeletal channels, in addition to the well documented lower affinity induction of prolonged substates. Transient activity was (a) enhanced with 0.2-10 nM toxin and (b) depressed by >50 nM toxin. The toxin at >1 nM enhanced Ca 2؉ release from SR in a manner consistent with two independent activation processes. The effects of the toxin on transient activity, as well as the toxin-induced substate, were independent of cytoplasmic Ca 2؉ or Mg 2؉ concentrations or the presence of adenine nucleotide and were seen in diisothiocyanostilbene-2,2-disulfonic acid-modified channels. Peptide A activated skeletal and cardiac channels with 100 nM cytoplasmic Ca 2؉ and competed with Imperatoxin A in the high affinity enhancement of transient channel activity and Ca 2؉ release from SR. In contrast to transient activity, prolonged substate openings induced by the toxin were not altered in the presence of peptide A. The results suggest that Imperatoxin A has three independent actions on ryanodine receptor channels and competes with peptide A for at least one action. Excitation-contraction (EC)1 coupling is the process that facilitates Ca 2ϩ release from the sarcoplasmic reticulum (SR) of muscle fibers following depolarization of the surface/transverse (t-) tubule membrane. A protein-protein interaction between the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR) underlies EC coupling in skeletal muscle. The DHPR L-type Ca 2ϩ channel in the t-tubule membrane detects surface depolarization and transmits a signal to the RyR channel in the SR via an interaction between the cytoplasmic domains of the two proteins. The interacting region of the DHPR is located between the second and third transmembrane repeats in the ␣1

Multiple actions of φ-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins

“…2) and maintained for >2 ms (and not an artifact of filtering)]. Although substate openings of RyRs occur constitutively (21), the number and duration of openings increased with higher LITX concentrations ( Fig. 2 A-F).…”

“…Low toxin concentrations increased the probability of full channel opening, and concentrations ∼100-fold higher inhibited the occurrence of the fully open state while inducing submaximal conductance states in single channel currents. These multiple actions are also exhibited by the scorpion calcine family toxin IpTxA, which increases full RyR openings at low nM concentrations and induces prolonged substates at low μM concentrations (21). Despite the absence of significant sequence homology (Fig.…”

“…At lower concentrations both toxins increase full openings of the RyR, and at higher concentrations they inhibit full openings and induce openings to subconductance levels and reduce the number of full conductance openings. We have previously suggested that the different actions are attributed to the toxins binding at different sites on the RyR, with binding at a high-affinity site mediating the increase in full openings and the induction of subconductance states evoked upon binding to a lower-affinity site (21). Examination of the 3D structure of LITX revealed that the W36 side chain is exposed on the surface of the molecule and not buried in the peptide core as would be expected for a hydrophobic residue.…”

“…This apparent incongruity is believed to be due to the existence of two or more ligand-binding sites on RyRs. It is thought IpTxA binds at a highaffinity site causing activation, whereas subconductance states arise on occupancy of lower-affinity sites (21). Other calcines, such as maurocalcine and hadrucalcin, demonstrate similar activities (22,23).…”

“…Originally isolated from the scorpion Pandinus imperator, imperatoxin A (IpTxA) is the best characterized of the calcines, with low μM IpTxA concentrations inducing RyR substates and inhibiting full single channel current opening. Interestingly, low nM IpTxA concentrations activate RyR by increasing transient activity and the probability of full channel openings (20,21). This apparent incongruity is believed to be due to the existence of two or more ligand-binding sites on RyRs.…”