Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation

Abstract

Channel adaptation is a fundamental feature of sarcoplasmic reticulum calcium release channels (called ryanodine receptors, RyRs). It permits successive increases in the intracellular concentration of calcium (Ca2+) to repeatedly but transiently activate channels. Adaptation of RyRs in the absence of magnesium (Mg2+) and adenosine triphosphate is an extremely slow process (taking seconds). Photorelease of Ca2+ from nitrophenyl-EGTA, a photolabile Ca2+ chelator, demonstrated that RyR adaptation is rapid (milliseconds) in canine heart muscle when physiological Mg2+ concentrations are present. Phosphorylation of the RyR by protein kinase A increased the responsiveness of the channel to Ca2+ and accelerated the kinetics of adaptation. These properties of the RyR from heart may also be relevant to other cells in which multiple agonist-dependent triggering events regulate cellular functions.

Fig. 1

RyR activity in the presence of a constant [Ca²⁺] and during a transient increase of the [Ca²⁺] in the absence of other modulators. Single channel openings are shown as upward deflections in all figures. The charge carrier is Cs⁺ and it flows from the luminal (trans) to the cytosolic (cis) side of the channel. Holding potential = −40 mV. (A) Continuous records of stabl activity of a single cardiac RyR channel. Concentration of free Ca²⁺ was 1 µM. (B) The same channel after buffering [Ca²⁺] to 100 nM (NP-EGTA, 1.5 mM; CaCl₂, 1.02 mM). Traces with asterisks (*) are expansions of segments in (A) and (C) marked with the corresponding symbols. (C) Correlation of an increase of RyR activity with a slow increase in the [Ca²⁺]. A train of low-power UV flashes was applied at a frequency of 10 Hz (arrows). The [Ca²⁺] in the vicinity of the channel was measured with a Ca²⁺ electrode positioned in the path of the light beam, about 0.2 mm in front of the bilayer aperture. (D) Relation of RyR activity to [Ca²⁺]. Continuous records in (A), (B), and (C) were divided into intervals of 500 ms; P, in each interval is plotted as a bar of length 0 to 1. Toptraces show the timecourse of the [Ca²⁺1 change near the bilayer surface (not bath [Ca²⁺]. Traces a, b, and c correspond to the calibrated voltage signal from the Ca²⁺ electrode obtained durng the recording of single-channel activity shown in panels (A) through (C), respectively.

Fig. 2

Activation of a RyR by very fast changes of the [Ca²⁺]. The resting [Ca²⁺] was 0.1 µM in all traces. Calibrated step increases of [Ca²⁺] were achieved by varying the power output of the laser apparatus [Q-switch mode (18)]. Resting conditions were reestablished by stirring the cis (that is, cytoplasmic) chamber. The RyR openings were elicited by fast increases of [Ca²⁺] to 1 µM (A and B) or to 10 µM (C) produced by single ca. 7-ns light pulses. A 1 mM concentration of free Mg²⁺ (1.02 mM MgCl₂) was present in (B) and (C). Traces in all panels were recorded from the same channel. (D) Ensemble currents were generated by the sum of data sweeps (curve a, 18 sweeps; curve b, 16 sweeps; and curve c, 23 sweeps) and correspond to the single-channel records shown in (A), (B), and (C), respectively. The time course of the spontaneous decay of activity was best fit by a single exponential function. The time constants of adaptation were (curve a) 1.41 s, (curve b) 98 ms, and (curve c) 168 ms. The means ± SD for n = 4 experiments were (curve a) 1.52 + 0.2 s, (curve b) 107 + 16 ms, and (curve c) 154 ± 27 ms. (E) Amplitude and time course of the change in [Ca²⁺] in the microenvironment of the channel as measured simultaneously with a Ca²⁺ electrode during the course of the experiment.

Fig. 3

Effect of Mg²⁺ on the peak, plateau, and rate of adaptation of RyR activity. (A) Ensemble currents obtained in the presence of the indicated [Mg²⁺] (0 to 3000 µM). Photolysis of caged Ca²⁺ increased [Ca²⁺] from 0.1 to 10 µM in all cases. The number of data sweeps at each [Mg²⁺], obtained from two independent experiments, were (0 µM Mg²⁺) 49, (30 µM Mg²⁺) 38, (300 µM Mg²⁺) 28, and (3000 µM Mg²⁺) 46. (B) The rate constant of spontaneous decay of RyR activity, τ_adapt, is plotted against [Mg²⁺]. Data points were fitted with the equation τ_adapt = τ_adaptmax/[1 +([Mg²⁺]/K_0.5)ⁿ] where τ_adaptmax is the rate constant of adaptation in the absence of Mg²⁺, K_0.5 is the half-maximal [Mg²⁺] necessary to accelerate adaptation, and n is the Hill number.

Fig. 4

Modification of RyR kinetics by PKA-dependent phosphorylation. Single laser pulses produced a photorelease-dependent increase in [Ca²⁺] from 0.1 to 10 puM in all panels. (A) Activation of a single RyR in the presence of 3 mM ATP and 4 mM MgCl₂ (a concentration of free Mg²⁺ of ca. 1 mM). (B) Representative traces of the same channel taken ca. 1 min after addition of the catalytic subunit (1 µg/ml) of PKA to the cytosolic solution (25). (C) Ensemble currents generated by summing 17 sweeps (curve a) and 21 sweeps (curve b) corresponding to the single-channel traces shown in panels (A) and (B), respectively. In each case, activity peaked within 5 ms after photolysis and then spontaneously decayed with a τ_adapt = 187 ms (curve a) or 106 ms (curve b).