Imperatoxin A Induces Subconductance States in Ca(2+) Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

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 potential 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 applied 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 different from the ryanodine binding site.