Unitary Ca(2+) Current through Cardiac Ryanodine Receptor Channels under Quasi-Physiological Ionic Conditions

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca(2+) currents were monitored when 2–30 mM Ca(2+) was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca(2+) current (at 0 mV holding potential) and lumenal [Ca(2+)] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH(3)SO(3) concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca(2+); and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca(2+) (n > 6). Unitary Ca(2+) current was also defined in the presence of symmetrical [Mg(2+)] (1 mM) and low [Cs(+)] (5 mM). Under these conditions, unitary Ca(2+) current in 2 and 10 mM lumenal Ca(2+) was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs(+)] (50 mM), Mg(2+) (1 mM), and lumenal [Ca(2+)] (10 mM), unitary Ca(2+) current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs(+) and Mg(2+) on unitary Ca(2+) current were additive. These data demonstrate that physiological levels of monovalent cation and Mg(2+) effectively compete with Ca(2+) as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca(2+) is 2 mM, then our results indicate that unitary Ca(2+) current under physiological conditions should be <0.6 pA.