Ryanodine does not affect calcium current in guinea pig ventricular myocytes in which Ca²⁺ is buffered

Calcium current in mammalian ventricular muscle is altered in the presence of ryanodine. Previous studies performed on rat ventricular cells have shown a slowing of Ca²⁺ current inactivation and suggest the hypothesis that ryanodine, by reducing the release of Ca²⁺ from the sarcoplasmic reticulum, reduces the availability of Ca²⁺ for inactivation of Ca²⁺ current (Ca²⁺ dependent inactivation). Another hypothesis is that the effects of ryanodine on Ca²⁺ current are due to a mechanical connection of the ryanodine receptor with the L-type Ca²⁺ channel. To further test these hypotheses we examined the effect of ryanodine on Ca²⁺ current in single voltage-clamped guinea pig ventricular myocytes that contained Ca²⁺ indicator and Ca²⁺ buffer. We used fura 2 (pentapotassium salt) to confirm that the ryanodine we used was capable of abolishing Ca²⁺ release from the sarcoplasmic reticulum during the period in which it was present. We perfused the cells with 10 mM EGTA to block changes in intracellular Ca²⁺ concentration. In the absence of internal EGTA, Ca²⁺ currents displayed biexponential inactivation and Ca²⁺ dependent inactivation (steady-state inactivation curves turned up at positive potentials). Inactivation was slowed by ryanodine at 10 μM. In cells perfused internally with EGTA, however, ryanodine had no effects, and steady-state inactivation curves were not shifted to the right. We conclude that, in guinea pig ventricular myocytes, the effects of ryanodine on Ca²⁺ current are mediated by Ca²⁺ and thus the effects of ryanodine do not provide a basis on which to postulate a physical connection between the L-type Ca²⁺ channel and the ryanodine receptor (sarcoplasmic reticulum Ca²⁺ release channel).