Ca²⁺ release-induced inactivation of Ca²⁺ current in rat ventricular myocytes: Evidence for local Ca²⁺ signalling

1. Inactivation of Ca²⁺ current (I(Ca)) induced by Ca²⁺ release from sarcoplasmic reticulum (SR) was studied in single rat ventricular myocytes using whole-cell patch-clamp and indo-1 fluorescence measurement techniques. 2. Depolarizing pulses to 0 mV elicited large Ca²⁺ transients and I(Ca) with biexponential inactivation kinetics. Varying SR Ca²⁺ loading by a 20 s pulse of caffeine showed that the fast component of I(Ca) inactivation was dependent on the magnitude of Ca²⁺ release. 3. Inactivation of I(Ca) induced by Ca²⁺ release was quantified, independently of voltage and Ca²⁺ entry, using a function termed fractional inhibition of I(Ca)(FI(Ca)). The voltage relation of FI(Ca) had a negative slope, resembling that of single-channel Ca²⁺ current (i(Ca)) rather than the bell-shaped current-voltage (I-V) relation of macroscopic I(Ca) and Ca²⁺ transients. 4. Intracellular dialysis of myocytes with 10 mM EGTA (150 nM free [Ca²⁺]) had no effect on I(Ca) inactivation induced by Ca²⁺ release, despite abolition of Ca²⁺ transients and cell contraction. Dialysis with 3 or 10 mM BAPTA (180 nM free [Ca²⁺]) attenuated FI(Ca) in a concentration-dependent manner, with greater inhibition at positive than at negative potentials, consistent with more effective buffering of Ca²⁺ microdomains of smaller i(Ca). 5. Spatial profiles of [Ca²⁺] near an opened Ca²⁺ channel were simulated. [Ca²⁺] reached submillimolar levels at the mouth of the channel, and dropped steeply as radial distance increased. At any given distance from the channel, [Ca²⁺] was higher at negative than at positive potentials. The radii of Ca²⁺ microdomains were significantly reduced by 3 or 10 mM BAPTA, but not by 10 mM EGTA. 6. In conclusion, the distinctive voltage dependence and susceptibility of Ca²⁺ release-induced I(Ca) inactivation to fast and slow Ca²⁺ buffers suggests that the process is mediated through local changes of [Ca²⁺] in the vicinity of closely associated Ca²⁺ channels and ryanodine receptors.