J. Gen. Physiol., Volume 111, Number 5, May 1, 1998 679-690

Imperatoxin A Induces Subconductance States in Ca²⁺ 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

Single-channel and [³H]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 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 [³H]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 Ca²⁺ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.

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