IMPERATOXIN-A INDUCES SUBCONDUCTANCE STATES IN CA2-MUSCLE( RELEASE CHANNELS (RYANODINE RECEPTORS) OF CARDIAC AND SKELETAL)

Single-channel and [H-3]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 Ca2+ release channels (CRCs). Single channel currents from purified CRCs incorporat ed into planar lipid bilayers were recorded in 250 mM KCl media. Addit ion of IpTx(a) in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanod ine receptor isoforms. The substates displayed a slightly rectifying c urrent-voltage relationship. The chord conductance at -40 mV was simil ar to 43% of the full conductance, whereas it was similar to 28% at a holding potential of +40 mV. The substate formation by IpTx(a) was vol tage and concentration dependent. Analysis of voltage and concentratio n dependence and kinetics of substate formation suggested that IpTx(a) reversibly binds to the CRC at a single site in the voltage drop acro ss 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 dissoc iation decreased e-fold per +25 mV applied holding potential. The effe ctive valence of the reaction leading to the substate was similar to 1 .5. The IpTx(a) binding site was calculated to be located at similar t o 23% of the voltage drop from the cytosolic side. IpTx(a) induced sub states in the ryanodine-modified skeletal CRC and increased or reduced [H-3]ryanodine binding to sarcoplasmic reticulum vesicles depending o n the level of channel activation. These results suggest that IpTx(a) induces subconductance states in skeletal and cardiac muscle Ca2+ rele ase channels by binding to a single, cytosolically accessible site dif ferent from the ryanodine binding site.