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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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Activation of the RyR channel by rapid Ca2+ spikes produced by flash photolysis of DM-nitrophen measured at three different bandwidths (left to right: 2, 5, and 10 kHz, respectively). (A, top) Time course of the reconstructed calcium spikes applied to the bilayer. (Middle) Sets of representative single channel records measured at +40 mV. The flash was applied at t = 0 ms (dotted lines). (Bottom) Ensemble currents constructed from 32–64 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom. (B) Exponential fits to the rising phase of the ensemble Po (expanded scale). Only every third point of Po is plotted for clarity (○). The continuous lines were obtained by fitting the data by the function:Po=Pmax1−e−tτana,where τa was 0.22 ± 0.01, 0.10 ± 0.01, and 0.09 ± 0.02 ms and n was 1.4 ± 0.4, 3.0 ± 0.5, and 2.8 ± 0.9 for 2, 5, and 10 kHz bandwidths, respectively. (C) The probability density of first latency (bars), the respective cumulative first latency distributions of channel openings (•), and open probability (dashed lines; same curve as ○ in B) at 2, 5, or 10 kHz.
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Figure 2: Activation of the RyR channel by rapid Ca2+ spikes produced by flash photolysis of DM-nitrophen measured at three different bandwidths (left to right: 2, 5, and 10 kHz, respectively). (A, top) Time course of the reconstructed calcium spikes applied to the bilayer. (Middle) Sets of representative single channel records measured at +40 mV. The flash was applied at t = 0 ms (dotted lines). (Bottom) Ensemble currents constructed from 32–64 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom. (B) Exponential fits to the rising phase of the ensemble Po (expanded scale). Only every third point of Po is plotted for clarity (○). The continuous lines were obtained by fitting the data by the function:Po=Pmax1−e−tτana,where τa was 0.22 ± 0.01, 0.10 ± 0.01, and 0.09 ± 0.02 ms and n was 1.4 ± 0.4, 3.0 ± 0.5, and 2.8 ± 0.9 for 2, 5, and 10 kHz bandwidths, respectively. (C) The probability density of first latency (bars), the respective cumulative first latency distributions of channel openings (•), and open probability (dashed lines; same curve as ○ in B) at 2, 5, or 10 kHz.

Mentions: We recorded single RyR channel activity in response to such brief free Ca2+ stimuli (Fig. 2 and Table ). The required temporal resolution was achieved by recording at a sampling rate of 100 kHz and cut-off filter setting ≥5 kHz. Before the flash, the channels exhibited essentially no activity. The channels responded to the Ca2+ stimulus in ∼25% of the episodes. The activity evoked by DMN photolysis consisted mostly of single openings, after which the channel stayed closed until the end of the episode (Fig. 2 A). To quantify the time course of channel activity, at least 32 single channel records obtained from an individual channel were combined to generate ensemble averages (Fig. 2 A, bottom).


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

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

Activation of the RyR channel by rapid Ca2+ spikes produced by flash photolysis of DM-nitrophen measured at three different bandwidths (left to right: 2, 5, and 10 kHz, respectively). (A, top) Time course of the reconstructed calcium spikes applied to the bilayer. (Middle) Sets of representative single channel records measured at +40 mV. The flash was applied at t = 0 ms (dotted lines). (Bottom) Ensemble currents constructed from 32–64 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom. (B) Exponential fits to the rising phase of the ensemble Po (expanded scale). Only every third point of Po is plotted for clarity (○). The continuous lines were obtained by fitting the data by the function:Po=Pmax1−e−tτana,where τa was 0.22 ± 0.01, 0.10 ± 0.01, and 0.09 ± 0.02 ms and n was 1.4 ± 0.4, 3.0 ± 0.5, and 2.8 ± 0.9 for 2, 5, and 10 kHz bandwidths, respectively. (C) The probability density of first latency (bars), the respective cumulative first latency distributions of channel openings (•), and open probability (dashed lines; same curve as ○ in B) at 2, 5, or 10 kHz.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230654&req=5

Figure 2: Activation of the RyR channel by rapid Ca2+ spikes produced by flash photolysis of DM-nitrophen measured at three different bandwidths (left to right: 2, 5, and 10 kHz, respectively). (A, top) Time course of the reconstructed calcium spikes applied to the bilayer. (Middle) Sets of representative single channel records measured at +40 mV. The flash was applied at t = 0 ms (dotted lines). (Bottom) Ensemble currents constructed from 32–64 individual episodes. The vertical calibration denotes 20 and 1 pA for the center and bottom. (B) Exponential fits to the rising phase of the ensemble Po (expanded scale). Only every third point of Po is plotted for clarity (○). The continuous lines were obtained by fitting the data by the function:Po=Pmax1−e−tτana,where τa was 0.22 ± 0.01, 0.10 ± 0.01, and 0.09 ± 0.02 ms and n was 1.4 ± 0.4, 3.0 ± 0.5, and 2.8 ± 0.9 for 2, 5, and 10 kHz bandwidths, respectively. (C) The probability density of first latency (bars), the respective cumulative first latency distributions of channel openings (•), and open probability (dashed lines; same curve as ○ in B) at 2, 5, or 10 kHz.
Mentions: We recorded single RyR channel activity in response to such brief free Ca2+ stimuli (Fig. 2 and Table ). The required temporal resolution was achieved by recording at a sampling rate of 100 kHz and cut-off filter setting ≥5 kHz. Before the flash, the channels exhibited essentially no activity. The channels responded to the Ca2+ stimulus in ∼25% of the episodes. The activity evoked by DMN photolysis consisted mostly of single openings, after which the channel stayed closed until the end of the episode (Fig. 2 A). To quantify the time course of channel activity, at least 32 single channel records obtained from an individual channel were combined to generate ensemble averages (Fig. 2 A, bottom).

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.

Show MeSH
Related in: MedlinePlus