679 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/98/05/679/12 $2.00 Volume 111 May 1998 679–690 http://www.jgp.org Imperatoxin A Induces Subconductance States in Ca 2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle Ashutosh Tripathy,* Wolfgang Resch,* Le Xu,* Hector H. Valdivia, ‡ and Gerhard Meissner* From the *Department of Biochemistry and Biophysics, and Department of Physiology, University of North Carolina, Chapel Hill, North Carolina 27599; and ‡ Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706 abstract Single-channel and [ 3 H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTx a ), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca 2+ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTx a in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at -40 mV was 43% of the full conductance, whereas it was 28% at a holding poten- tial of +40 mV. The substate formation by IpTx a was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTx a reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTx a binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV ap- plied holding potential. The effective valence of the reaction leading to the substate was 1.5. The IpTx a binding site was calculated to be located at 23% of the voltage drop from the cytosolic side. IpTx a induced substates in the ryanodine-modified skeletal CRC and increased or reduced [ 3 H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTx a induces subconductance states in skeletal and cardiac muscle Ca 2+ release channels by binding to a single, cytosolically accessible site differ- ent from the ryanodine binding site. key words: sarcoplasmic reticulum • imperatoxin activator • substates • scorpion toxin • [ 3 H]ryanodine binding introduction Ca 2+ release channels (CRCs), 1 also known as ryano- dine receptors (RyRs), mediate the release of Ca 2+ from an intracellular membrane compartment, the sar- coplasmic reticulum (SR), into the cytoplasm in stri- ated muscle cells. The CRC has been purified as a 30 S protein complex and shown to be composed of four large RyR polypeptides of 5,000 amino acid residues and four immunophilins (FK506 binding protein) of 100 amino acid residues each. Three tissue-specific isoforms of RyR have been identified. The conduc- tances and pharmacological properties of the three iso- forms are, to a large extent, similar (for reviews, see Meissner, 1994; Sutko and Airey, 1996). Traditionally, venom from many poisonous snakes, lizards, and scorpions has been a rich source of peptide toxins that target various voltage- and ligand-gated channels including the CRCs (Furukawa et al., 1994; Morrissette et al., 1995, 1996). Recently, from the venom of the African scorpion Pandinus imperator, two factors, imperatoxin activator (IpTx a ) and imperatoxin inhibitor (IpTx i ), were isolated that selectively activated and inhibited, respectively, the CRCs (Valdivia et al., 1992; El-Hayek et al., 1995). IpTx a is a 33 amino acid peptide that activated the skeletal CRC in [ 3 H]ryano- dine binding and single channel studies. It did not ex- ert a significant effect on cardiac CRC (Valdivia et al., 1992; El-Hayek et al., 1995; Zamudio et al., 1997a). IpTx i has been shown to be a heterodimeric protein (subunits of 108 and 27 amino acid residues covalently linked by a disulfide bond) with lipolytic action, and it inhibited both skeletal and cardiac CRCs by generating a lipid product (Valdivia et al., 1992; Zamudio et al., 1997b). Here, we show that IpTx a induces voltage- and con- centration-dependent subconductance states in both skeletal and cardiac CRCs when added to the cytosolic side. Analysis of voltage and concentration dependence and stochastic analysis of the events suggests that the in- duction of subconductance states corresponds to re- Address correspondence to Ashutosh Tripathy, Ph.D., Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260. FAX: 919-966-2852; E-mail: tripathy @med.unc.edu 1 Abbreviations used in this paper: CRC, Ca 2+ release channel; IpTx a , imperatoxin activator; RyR, ryanodine receptor; SR, sarcoplasmic reticulum. Downloaded from http://rupress.org/jgp/article-pdf/111/5/679/1190778/gp-7617.pdf by guest on 03 September 2022