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 r esponse of the L-type calcium current (I(Ca)) of guinea-pig ventricula r cells to a rapid increase in the free intracellular calcium concentr ation ([Ca2+]i). 2. When 2 mm nitr-5 or 3 mm DM-nitrophen was loaded i nto 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 blo ck was reduced by increasing the pre-flash [Ca2+]i and enhanced by add ing high concentrations of Ca2+ chelators to the pipette-filling solut ion. 3. The photolysis-induced block was not suppressed in the presenc e 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 wit h various levels of pre-flash [Ca2+]i, peak I(Ca) was approximately do ubled 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 exponentia l 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 w as 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 extr acellular Ca2+ with Ba2+ significantly suppressed the flash-induced po tentiation. 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 t he pipette. Potentiation of Ba was largely blocked in Ca2+-depleted ce lls when CaCl2 was omitted from the pipette. 7. When I(Ca) was activat ed 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-indu ced potentiation was best fitted by two exponentials; one was similar to the single component seen at 0.05 Hz and the other was approximatel y one order of magnitude faster. The contribution of the faster compon ent was positively correlated to the stimulation frequency. 8. The fla sh-induced potentiation of I(Ca) was suppressed in the presence of a s upramaximal 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 ki nase inhibitors H-7 and Rp-cAMP-S had no significant effect on the pho tolysis-induced potentiation, but the non-hydrolysable ATP analogue AM P-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 conclud e 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 i s probably caused by a direct interaction between the photoreleased Ca 2+ and the Ca2+ channel. The Ca2+ dependence of the photoinduced poten tiation is explained in terms of a model with two binding sites for Ca 2+; 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.