Unitary Ca2+ current through mammalian cardiac and amphibian skeletal muscle ryanodine receptor Channels under near-physiological ionic conditions

Abstract

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1-30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (approximately 55%) in mammalian and amphibian channels. Two amplitudes, differing by approximately 35%, were found in amphibian channel studies, probably corresponding to alpha and beta RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (approximately 40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.

Figure 1.

Unitary Ca²⁺ current of amphibian (A) or mammalian (B) RyR channels in the absence of competing ions. In this and the following figures currents were recorded at 0 mV in the presence of caffeine (10 or 5 mM, see materials and methods), and filtered at 1 kHz. Open events are shown as downward current deflections. Records in the figure were at 5 mM [Ca²⁺]_L. The open state is marked by a filled arrowhead. The closed state is marked by an open arrowhead. Corresponding all-points histograms reveal channel currents of <3 pA.

Figure 2.

Mammalian RyR2 channel current is attenuated by physiological levels of K⁺ or Mg²⁺. [Ca²⁺]_L was 2 mM. All data were collected from the same single channel. (A) Representative control recordings and all-points histogram. (B) Current after symmetrical addition of 150 mM KCl. (C) Current after solutions were changed to contain symmetrical 1 mM Mg²⁺.

Figure 3.

Amphibian skeletal muscle RyR channel current is attenuated by physiological levels of K⁺ or Mg²⁺. [Ca²⁺]_L was 4 mM. (A) Representative recording and corresponding all-points histogram. (B) Current after symmetrical addition of 150 mM KCl and 1 mM Mg²⁺.

Figure 4.

Current-voltage relationships of a RyR channel of amphibian skeletal muscle in the presence of different KCl concentrations. Squares, 50 mM KCl; open circles, 100 mM; filled circles, 200 mM. All data were collected on the same channel while salt concentration was increased by serial symmetrical additions. At each salt concentration data were well fit by a straight line. The slope conductances were 258, 613, and 721 pS for the 50, 100, and 200 mM datasets, respectively. Current reversed very close to 0 mV in all cases (inset).

Figure 5.

Different current amplitudes suggest the presence of two classes of channels. (A) Example multilevel recording acquired under control conditions (no competing ions). [Ca²⁺]_L was 20 mM. Cytosolic [Ca²⁺] was 10 μM. (B) Corresponding all points histogram. Note peaks at two different individual amplitudes and their sum.

Figure 6.

Distribution of current amplitudes in frog skeletal channels. The curve fits are either one or the sum of two Gaussian functions of current I (i.e., a₁ exp(−(I − μ₁)²/2σ₁ ²). (A) Average values from bilayer experiments that featured two different current amplitudes, as illustrated in Fig. 5. The two histograms plot separately high (filled bars) and low (hatched bars) amplitudes at 20 mM [Ca²⁺]_L. The fit parameters are μ₁ = −4.19 pA, σ₁ = 0.539, μ₂ = −3.22 pA and σ₂ = 1.01. (B). The dotted line represents the Gaussian fit of the high current amplitudes presented in part A. The crosshatched bars represent amplitudes in bilayer experiments that had one level, or multiple levels at equal intervals. The solid curve represents a constrained fit with the sum of two Gaussians: a₁ = 0.71, μ₁ = −4.14 pA, σ₁ = 0.65, a₂ = 0.41, μ₂ = −3.22 pA and σ₂ = 1.10.

Figure 7.

Unitary Ca²⁺ current of amphibian and mammalian RyR channels in the presence of competing ions. (A) Schematic representation of the four different salt conditions examined. Filled triangle, control (Ca²⁺ in the absence of other permeant ions). Open circle, symmetric 1 mM Mg²⁺. Filled circle, symmetric 150 mM K⁺. Half-open circle, symmetric 1 mM Mg²⁺ and 150 mM K⁺. (B) Unitary Ca²⁺ current carried by the mammalian RyR2 channel in each condition, plotted vs. [Ca²⁺]_L. The curves are fits obtained using equation 1 with parameters B and i_MAX. The B values are 0.45, 0.33, 0.37 and 0.32 mM^-1 for the control, Mg-only, K-only, and Mg-K datasets, respectively. The corresponding i_MAX values are 4.44, 3.42, 2.87 and 2.76 pA. (C) Unitary Ca²⁺ current (at 0 mV) carried by the amphibian skeletal muscle RyR in each test condition, plotted versus [Ca²⁺]_L. Again, the curves were fit using Eq. 1. The B values are 0.51, 0.24, 0.17, and 0.17 for the control, Mg-only, K-only, and Mg-K datasets, respectively. The corresponding i_MAX values are 4.25, 3.79, 3.56, and 3.26 pA. (D) The mammalian and amphibian Mg²⁺ and KCl datasets replotted on an expanded scale. Open triangles, currents calculated with the 4-barrier model (see methods) assuming symmetric 1 mM Mg²⁺ and 150 mM K⁺. The dotted line represents the published data of Mejía-Alvarez et al. (1999) collected in symmetrical 150 mM CsCH₃SO₃.

Figure 8.

Influence of elevated Mg²⁺ on Ca²⁺ current of frog RyR channel. Experiments at 10 mM [Ca²⁺]_L. (A) Current in the presence of symmetric 0, 5 or 10 mM Mg²⁺ (top to bottom). (B) Marked segments of records in A on an expanded time scale. (C) Squares, average currents (± SEM; n = 3–5) in different symmetrical Mg²⁺ concentrations. Line, fit with Eq. 1 to predictions of the 4-barrier model. Filled circle, measured current at 1 mM [Mg²⁺] and 150 mM KCl. Open circle, corresponding prediction of the 4-barrier model.