Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37ºC, dialysed internally with cAMP-free and cAMP-containing solutions

Abstract

In many mammalian tissue types an integral membrane protein – the sodium/calcium (Na/Ca) exchanger – plays a key role in intracellular Ca homeostasis, and evidence suggests that Na/Ca exchange function can be modulated by cAMP-dependent phosphorylation. External Nickel (Ni) ions are used widely to inhibit the exchange but little is known about the mode of Ni action. In guinea-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by external Ni under phosphorylated (cells dialysed with cAMP) and non-phosphorylated conditions. Ventricular myocytes were isolated from adult guinea-pig hearts, recordings were made at 37°C using the whole-cell patch clamp technique. Internal and external solutions were used which allowed Na/Ca exchange current (INaCa) to be measured during a descending voltage ramp protocol (+80 to –120 mV) applied from a holding potential of –40 mV. The application of 10 mM Ni caused a maximal block of INaCasince inhibition was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superfused externally. Kinetics of Ni-block of INaCawere assessed using applications of different external [Ni] to cells dialysed internally with cAMP-free and 100 μM cAMP-containing solutions. At +60 mV, Ni inhibited INaCain cells dialysed with a cAMP-free solution with a dissociation constant (KD) of 0.29 ± 0.03 mM and the data were fitted with a Hill coefficient of 0.89 ± 0.07 (n= 9 cells). In cells dialysed with 100 μM cAMP the exchange was inhibited by Ni with a KDof 0.16 ± 0.05 mM, the Hill coefficient was 0.82 ± 0.16 (n= 6–7 cells). The KDand Hill coefficient values obtained in cells dialysed with cAMP-free and cAMP-containing solutions were not significantly different. Inhibition of INaCaby Ni did not appear to be voltage-dependent, was maximal within 3–4 s of application and was rapidly reversible. With cAMP-free internal dialysate, inhibition was ‘mixed’ showing competition with external Ca and a degree of non-competitive block. With 100 μM cAMP the inhibition appeared to be more non-competitive. We conclude that, under these experimental conditions, a concentration of external Ni of 10 mM is sufficient to produce maximal inhibition of INaCain guinea-pig cardiac cells.