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Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli.

Zahradníková A, Zahradník I, Györke I, Györke S - J. Gen. Physiol. (1999)

Bottom Line: To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen.These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings.They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic 83334, USA.

ABSTRACT
The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.

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Simulation of RyR responses to Ca2+ spikes using RyR gating models with one or four Ca2+ binding sites. (A) The gating schemes for Model 1Ca (left) and Model 4Ca (right; the rate constants are given in Table ). (B, top) The time course of the calcium spikes. (Middle) Sets of representative simulated single channel episodes filtered at 10 kHz. (Bottom) Ensemble currents constructed from 128 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom, respectively. The dotted lines denote the time of application of the Ca2+ spikes. (C) The probability density of first latency (bars) along with the respective cumulative first latency distributions (•) of channel openings. The ensemble open probabilities are overlaid (solid lines).
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Figure 6: Simulation of RyR responses to Ca2+ spikes using RyR gating models with one or four Ca2+ binding sites. (A) The gating schemes for Model 1Ca (left) and Model 4Ca (right; the rate constants are given in Table ). (B, top) The time course of the calcium spikes. (Middle) Sets of representative simulated single channel episodes filtered at 10 kHz. (Bottom) Ensemble currents constructed from 128 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom, respectively. The dotted lines denote the time of application of the Ca2+ spikes. (C) The probability density of first latency (bars) along with the respective cumulative first latency distributions (•) of channel openings. The ensemble open probabilities are overlaid (solid lines).

Mentions: To further characterize the activation of RyRs by Ca2+ spikes, we measured RyR activity in response to laser flashes of different intensities. Fig. 4A–C, shows channel responses to laser flashes of low, intermediate, and high intensity along with the corresponding calculated free [Ca2+] spikes in a representative experiment. In this experiment, the amplitude of the Ca2+ spike was estimated to be 9.3, 18.3, and 27.4 μM for low, intermediate, and high intensity pulses, respectively. The Ca2+ spikes decayed with time constants of 0.17, 0.18, and 0.20 ms, respectively. Ca2+ was elevated to over 5 μM for 0.13, 0.27, and 0.34 ms, and to over 1 μM for 0.4, 0.6, and 0.7 ms, respectively. As can be seen, low-intensity flashes caused channel openings only in relatively few occasions (peak Po ∼ 0.06); increasing flash energy increased the probability of activation (peak Po ∼ 0.25 and 0.50, respectively). Interestingly, in all cases the responses were composed of isolated openings with a similar duration. The time constants of activation, determined by fitting single exponential association function raised to the power n to the ensemble averages, progressively decreased with increasing the energy of the laser pulse (τa = 0.27, 0.09, and 0.07 ms; na = 3.5, 2.5, and 2.4, respectively; Fig. 5, D–F). Similar results were obtained in five other experiments. These results are summarized in Fig. 6 F, which plots the peak Po of the channel as a function of spike amplitude. The [Ca2+] dependence of Po could be described by with a KCa value of 29 ± 1 μM and an apparent Hill slope of 2.5 ± 0.2. The high values of the activation exponent and of the Hill slope further indicate that activation of the RyR channel requires binding of several calcium ions.


Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli.

Zahradníková A, Zahradník I, Györke I, Györke S - J. Gen. Physiol. (1999)

Simulation of RyR responses to Ca2+ spikes using RyR gating models with one or four Ca2+ binding sites. (A) The gating schemes for Model 1Ca (left) and Model 4Ca (right; the rate constants are given in Table ). (B, top) The time course of the calcium spikes. (Middle) Sets of representative simulated single channel episodes filtered at 10 kHz. (Bottom) Ensemble currents constructed from 128 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom, respectively. The dotted lines denote the time of application of the Ca2+ spikes. (C) The probability density of first latency (bars) along with the respective cumulative first latency distributions (•) of channel openings. The ensemble open probabilities are overlaid (solid lines).
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Related In: Results  -  Collection

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Figure 6: Simulation of RyR responses to Ca2+ spikes using RyR gating models with one or four Ca2+ binding sites. (A) The gating schemes for Model 1Ca (left) and Model 4Ca (right; the rate constants are given in Table ). (B, top) The time course of the calcium spikes. (Middle) Sets of representative simulated single channel episodes filtered at 10 kHz. (Bottom) Ensemble currents constructed from 128 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom, respectively. The dotted lines denote the time of application of the Ca2+ spikes. (C) The probability density of first latency (bars) along with the respective cumulative first latency distributions (•) of channel openings. The ensemble open probabilities are overlaid (solid lines).
Mentions: To further characterize the activation of RyRs by Ca2+ spikes, we measured RyR activity in response to laser flashes of different intensities. Fig. 4A–C, shows channel responses to laser flashes of low, intermediate, and high intensity along with the corresponding calculated free [Ca2+] spikes in a representative experiment. In this experiment, the amplitude of the Ca2+ spike was estimated to be 9.3, 18.3, and 27.4 μM for low, intermediate, and high intensity pulses, respectively. The Ca2+ spikes decayed with time constants of 0.17, 0.18, and 0.20 ms, respectively. Ca2+ was elevated to over 5 μM for 0.13, 0.27, and 0.34 ms, and to over 1 μM for 0.4, 0.6, and 0.7 ms, respectively. As can be seen, low-intensity flashes caused channel openings only in relatively few occasions (peak Po ∼ 0.06); increasing flash energy increased the probability of activation (peak Po ∼ 0.25 and 0.50, respectively). Interestingly, in all cases the responses were composed of isolated openings with a similar duration. The time constants of activation, determined by fitting single exponential association function raised to the power n to the ensemble averages, progressively decreased with increasing the energy of the laser pulse (τa = 0.27, 0.09, and 0.07 ms; na = 3.5, 2.5, and 2.4, respectively; Fig. 5, D–F). Similar results were obtained in five other experiments. These results are summarized in Fig. 6 F, which plots the peak Po of the channel as a function of spike amplitude. The [Ca2+] dependence of Po could be described by with a KCa value of 29 ± 1 μM and an apparent Hill slope of 2.5 ± 0.2. The high values of the activation exponent and of the Hill slope further indicate that activation of the RyR channel requires binding of several calcium ions.

Bottom Line: To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen.These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings.They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic 83334, USA.

ABSTRACT
The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.

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