CA2+-DEPENDENT BLOCK AND POTENTIATION OF L-TYPE CALCIUM CURRENT IN GUINEA-PIG VENTRICULAR MYOCYTES

1. The caged calcium compound nitr-5 has ben used to investigate the response of the L-type calcium current (I(Ca)) of guinea-pig ventricular cells to a rapid increase in the free intracellular calcium concentration ([Ca2+]i). 2. When 2 mm nitr-5 or 3 mm DM-nitrophen was loaded into cells via a patch pipette and photolysed during the decay phase of I(Ca), a partial block of the current developed within 75 ms. The block was reduced by increasing the pre-flash [Ca2+]i and enhanced by adding high concentrations of Ca2+ chelators to the pipette-filling solution. 3. The photolysis-induced block was not suppressed in the presence of isoprenaline, suggesting a direct action of Ca2+ on the channels rather than a mechanism involving channel phosphorylation. 4. The most prominent effect of nitr-5 photolysis was a slow potentiation of I(Ca). When I(Ca) was activated at frequencies between 0.05 and 0.7 Hz with various levels of pre-flash [Ca2+]i, peak I(Ca) was approximately doubled in amplitude following photolysis. 5. At a stimulation frequency of 0.05 Hz, when nitr-5 was the only chelator present in the pipette, the time course of the potentiation was fitted by a single exponential with a time constant (tau(P)) of 2.7 min. When 1 mm CaCl2 was added to the pipette-filling solution, the time course of the potentiation was slowed (tau(P) = 6 min), although its amplitude was unchanged. With 12 mm BAPTA (a calcium chelator) added instead of CaCl2, the response was accelerated (tau(P) = 1.7 min). 6. Equimolar substitution of extracellular Ca2+ with Ba2+ significantly suppressed the flash-induced potentiation. The time course of the potentiation of the barium current, I(Ba) (tau(P) = 1.9 min) was similar to that of I(Ca) with BAPTA in the pipette. Potentiation of Ba was largely blocked in Ca2+-depleted cells when CaCl2 was omitted from the pipette. 7. When I(Ca) was activated at frequencies of greater-than-or-equal-to 0.1 Hz, with 1 mm CaCl2 added to the nitr-5 (2 mm) in the pipette, the onset of the flash-induced potentiation was best fitted by two exponentials; one was similar to the single component seen at 0.05 Hz and the other was approximately one order of magnitude faster. The contribution of the faster component was positively correlated to the stimulation frequency. 8. The flash-induced potentiation of I(Ca) was suppressed in the presence of a supramaximal concentration of the beta-adrenergic agonist isoprenaline. Stimulation of I(Ca) by isoprenaline was significantly reduced after augmentation of the current by photolysis of nitr-5. 9. The protein kinase inhibitors H-7 and Rp-cAMP-S had no significant effect on the photolysis-induced potentiation, but the non-hydrolysable ATP analogue AMP-PNP caused a greater than twofold increase in the potentiation when compared with ATP. The magnitude of the potentiation was dependent on the concentration of ATP added to the pipette solution. 10. We conclude that the response of I(Ca) to a rapid increase in [Ca2+]i in guinea-pig ventricular cells is complex. The immediate block of the current is probably caused by a direct interaction between the photoreleased Ca2+ and the Ca2+ channel. The Ca2+ dependence of the photoinduced potentiation is explained in terms of a model with two binding sites for Ca2+; one site is close to the channel mouth and triggers the response, while the other is inhibitory. The potentiation is not mediated by Ca2+-dependent phosphorylation, but it does involve a nucleotide.