Unitary Ca2+ current through cardiac ryanodine receptor channels under quasi-physiological ionic conditions

Abstract

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 Ca2+ currents were monitored when 2-30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at approximately 4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 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 Ca2+; and 3.3 +/- 0.4, 2.4 +/- 0. 2, and 1.63 +/- 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 +/- 0.1 and 1.52 +/- 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 +/- 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA.

Figure 7

Schematic representation of the electrostatic focussing diffusion model. Ca²⁺ ions diffuse in a spherically symmetrical manner, passing through a permeable, negatively charged sphere of radius r ₁ to an inner sphere of radius r ₀ (which represents the capture radius of the true pore). The ions reappear at r ₀ (having passed through the pore without resistance) and diffuse spherically into the cytosolic space, passing again through the charged sphere. A Donnan equilibrium potential difference exists across the outer boundary of the charged sphere, increasing [Ca²⁺] by a Nernst factor upon entering the sphere.

Figure 1

Unitary Ca²⁺ current in absence of competing ions. Single-channel current was recorded at 0 mV holding potential, in absence of monovalent ions and using 2 mM Ca²⁺ as current carrier. (A) Representative trace of the single-channel activity recorded under these conditions. Opening events are displayed as downward deflections. The continuous line represents the zero current level, while the dotted line represents the mean opening level. Current signal was filtered at 1 kHz. (Right) Total amplitude histogram obtained from the same experiment. Continuous lines were generated by fitting a double Gaussian function. The opening distribution was centered at −1.37 pA. (B) Single-channel activity recorded under conditions similar to A, before and after addition of 20 μM of ryanodine to the cytoplasmic side of the channel. Traces after ryanodine were filtered at 150 Hz to resolve the squared openings. (C) Single-channel activity recorded under control conditions and after addition of 5 μM ruthenium red to both sides of the channel.

Figure 2

Unitary Ca²⁺ current in presence of 150 mM Cs⁺ and without Mg²⁺. Single-channel activity recorded at 0 mV holding potential, using 2 mM Ca²⁺ as current carrier, and in the presence of 150 mM symmetrical Cs⁺. (A) Representative trace of the unitary current recorded under these conditions. Opening events are displayed as downward deflections. The continuous line represents the zero current level, while the dotted line indicates the mean opening level. The current signal was filtered at 300 Hz. (B) Total amplitude histogram, fitted by a sum of two gaussian functions, revealing an open channel current of 0.35 pA.

Figure 3

Effect of different [Cs⁺]'s on unitary Ca²⁺ current amplitude. Unitary Ca²⁺ current amplitude was measured at 0 mV, in the absence of Mg²⁺, in 5 (○; n = 6), 50 (•; n = 5), and 150 (▵; n = 3) mM Cs⁺. Experimental data were fitted by single rectangular hyperbolic functions.

Figure 4

Effect of different [Cs⁺]'s on unitary Ba²⁺ current amplitude. Unitary Ba²⁺ current amplitude was measured at 0 mV, in 5 (○; n = 6), 50 (•; n = 4), and 150 (▵; n = 3) mM Cs⁺. Experimental data were fitted by single rectangular hyperbolic functions.

Figure 5

Effect of Mg²⁺ concentration on unitary Ca²⁺ current amplitude. Ca²⁺ current amplitude was measured at 0 mV, in 2 (top), 5 (middle), and 10 (bottom) mM Ca²⁺, in the absence (left) and presence (right) of 1 mM Mg²⁺. Current signal was filtered at 1 kHz, except for the trace in 2 mM Ca²⁺ and 1 mM Mg²⁺, which was filtered at 300 Hz. Continuous lines indicate the zero current level and the dotted lines indicate the mean opening level. The corresponding values of current amplitude obtained from the total amplitude histograms are indicated below each trace.

Figure 6

Unitary Ca²⁺ current amplitude at different lumenal [Ca²⁺]'s in the simultaneous presence of Mg²⁺ and Cs⁺. (A) Unitary Ca²⁺ current amplitude at 0 mV was measured in the virtual absence of competing ions (0 mM Mg²⁺ and 5 mM Cs⁺, •, n = 6), with 1 mM symmetrical Mg²⁺ and 5 mM Cs⁺ (○, n = 5), and with 1 mM Mg²⁺ plus 50 mM symmetrical Cs⁺ (shaded triangles, n = 2). The results were plotted as a function of the lumenal Ca²⁺. For the first two conditions, the experimental data were fitted with a single rectangular hyperbolic function. For comparison, the hyperbolic function fitted to the data obtained in the absence of competing ions was scaled to the multi-ion data. (B) Representative current trace obtained at 0 mV, with 10 mM lumenal Ca²⁺, 10 μM cytoplasmic Ca²⁺, 1 mM symmetrical Mg²⁺, and 50 mM symmetrical Cs⁺. (C) The corresponding total amplitude histogram fitted with two gaussian functions reveals a mean opening level with an amplitude of ∼0.7 pA.