Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes - PubMed (original) (raw)

Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes

J S Sham et al. Proc Natl Acad Sci U S A. 1998.

Abstract

In heart, a robust regulatory mechanism is required to counteract the regenerative Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca2+ release by using a technique of imaging local Ca2+ release, or "Ca2+ spikes", at subcellular sites; and we tracked the kinetics of Ca2+ release triggered by L-type Ca2+ channels. At 0 mV, Ca2+ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca2+ channels with the Ca2+ channel agonist, FPL64176, did not prolong or trigger secondary Ca2+ spikes, even though two-thirds of the sarcoplasmic reticulum Ca2+ remained available for release. Latency of Ca2+ spikes coincided with the first openings but not with the reopenings of L-type Ca2+ channels. After an initial maximal release, even a multi-fold increase in unitary Ca2+ current induced by a hyperpolarization to -120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca2+ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca2+ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.

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Figures

Figure 1

Figure 1

FPL increases open probability, prolongs open duration, and prevents Ca2+ release-induced inactivation of Ca2+ channel. (A) Representative traces of unitary Ca2+ current recorded from a single channel patch at 0 mV, using 10 mM Ca2+ as the charge carrier in the presence of 10 μM FPL. (A Bottom) the ensemble averaged current of 925 sweeps. Notice the prolonged openings and reopenings during the pulse. (B) Open-time histogram of 1,907 opening events, bin width = 0.4 ms. The smooth line represents the best-fitted probability distribution function and τs denote the time constants. (C) Latency distribution of the first openings and reopenings of Ca2+ channel, bin width = 1 ms. Number of first openings is 637 and reopenings is 1,270. (D) Currents of the first opening (I1st) and reopenings (IR) of Ca2+ channels, reconstructed from idealized events. (A to D) Generated from the same patch. (E) Whole-cell ICa and Ca2+ transients in the presence of 3 μM FPL, before and during superfusion of 10 mM caffeine. (F) Inactivation of whole-cell ICa, quantified as I25 ms/Ipeak, before and during superfusion of 10 mM caffeine, in control and FPL-treated (n = 13) myocytes. ∗, Significant difference from control (P < 0.05).

Figure 2

Figure 2

Effect of FPL on SR Ca2+ release. (A) Ca2+ release immediately before (Left column) and during the first depolarizing pulse (Right column) after application of 10 μM FPL. From Top to Bottom: voltage protocols, confocal line-scan images, normalized spatially averaged OG-5N signals (F/F0), ICa, and superimposed peak normalized ICa (black traces) and SR Ca2+ release function (_f_r, red traces). Vertical and horizontal axes of line-scan images are axes of space and time, respectively. Smooth lines in the F/F0 panels represent _f_s and red lines in the Bottom panels represent _f_r generated by subtracting f_s from the ΔF/F0 traces. (B) Latency distribution of Ca2+ spikes (n = 376) elicited in 10 myocytes during the first and second pulses after application of FPL (10 μM). The red and blue lines are the scaled latency distributions of the first openings and reopenings, respectively, of Ca2+ channel of Fig. 1_C.

Figure 3

Figure 3

Caffeine-induced Ca2+ release during the maintained phase of ICa. (A Top to Bottom) voltage protocol, confocal line-scan image, spatially averaged OG-5N fluorescence signal, and the simultaneously measured ICa. FPL (10 μM) was applied 10 s before depolarization, and caffeine was rapidly injected onto the cell 40 ms after the onset of depolarization. The smooth line in the Middle panel represents _f_s. (B) Amount of SR Ca2+ released by ICa and caffeine quantified by integrating their respective release fluxes (_f_r) transients (n = 6).

Figure 4

Figure 4

Single channel current and SR Ca2+ release induced by depolarization and subsequent hyperpolarization. (A) Representative traces of single Ca2+ channel current recorded in the presence of 10 μM FPL, during depolarizing pulses (50 ms) to −30, 0, and +30 mV followed by hyperpolarizing steps to −120 mV. C, O, and T, mark the _i_Ca when the channel was closed, open, and open during hyperpolarization (tail opening), respectively. Notice the multi-fold step increase in _i_Ca upon hyperpolarization. (B) Representative line-scan images, spatially averaged OG-5N fluorescence signals and ICa simultaneously recorded in a myocyte. (C) Peak Ca2+ release fluxes elicited by a depolarizing pulse (to −30, 0, +30, or +60 mV, black bars) followed by a hyperpolarizing step (gray bars). (D) Cumulative Ca2+ release elicited by both depolarizing and hyperpolarizing pulses quantified by integrating _f_r. Bar graphs C and D are the averaged data from five cells.

Figure 5

Figure 5

Local Ca2+ release transients elicited at individual t-tubular-SR junctional sites in the presence of 10 μM FPL. (A) Confocal line-scan image (Upper) and Ca2+ spikes (Lower) elicited by a single depolarizing pulse to +30 mV for 50 ms, followed by repolarization to −60 mV. Numbers in the image indicate the spatial locations of the six individual release sites at which the Ca2+ spikes displayed in the Lower panel were recorded. (B) Scatter plot of amplitudes of depolarization-induced Ca2+ spikes vs. amplitudes of those triggered by the subsequent repolarization. The straight line is the least square linear regression of the data, r is the correlation coefficient, and n = 170.

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