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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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Defining the properties of the rapid Ca2+ spikes. (A) The reaction scheme of DMN complexation and photolysis (Ellis-Davies et al. 1996). The light-induced transitions of DMN into the activated states are assumed to be irreversible and instantaneous, and only a fraction of DMN (ΔDMN; 0.5–2% in our experiments) undergoes this reaction step. Other transitions in the scheme are reversible. (B) The increase in steady state Ca2+ after the flash plotted as a function of preflash steady state Ca2+ in five independent experiments. The total DMN concentration was always 3 mM. Different symbols denote different power settings of the laser flash. For a given flash energy the log–log plot of ΔCa2+ vs. Ca2+ was linear and was used to precalibrate flash energy in a given experiment. (C) The time course of a typical calcium spike reconstructed from the pre- and postflash steady state Ca2+ concentrations. The on and off portions of the calcium profile could be fitted by monoexponential functions, giving τon = 6.5 ± 1.5 μs and τoff = 106 ± 1 μs.
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Figure 1: Defining the properties of the rapid Ca2+ spikes. (A) The reaction scheme of DMN complexation and photolysis (Ellis-Davies et al. 1996). The light-induced transitions of DMN into the activated states are assumed to be irreversible and instantaneous, and only a fraction of DMN (ΔDMN; 0.5–2% in our experiments) undergoes this reaction step. Other transitions in the scheme are reversible. (B) The increase in steady state Ca2+ after the flash plotted as a function of preflash steady state Ca2+ in five independent experiments. The total DMN concentration was always 3 mM. Different symbols denote different power settings of the laser flash. For a given flash energy the log–log plot of ΔCa2+ vs. Ca2+ was linear and was used to precalibrate flash energy in a given experiment. (C) The time course of a typical calcium spike reconstructed from the pre- and postflash steady state Ca2+ concentrations. The on and off portions of the calcium profile could be fitted by monoexponential functions, giving τon = 6.5 ± 1.5 μs and τoff = 106 ± 1 μs.

Mentions: Fast changes of the Ca2+ concentration in the microenvironment of the reconstituted channel were performed by flash photolysis of DM-nitrophen (Calbiochem Corp.) as described previously (Györke and Fill 1993; Györke et al. 1994). Intense, 9-ns long UV laser flashes produced by a pulsed, frequency-tripled, Nd:YAG laser (Spectra-Physics) were applied through a fused silica fiber optics (450 μm diameter) positioned perpendicular to the bilayer surface (100 μm diameter) so that the whole volume between the fiber optics and the bilayer was illuminated evenly and instantaneously. The amplitude and time course of Ca2+ after the flash were determined from the concentration of total and free DMN and Ca2+, and from the proportion of DMN photolyzed during the flash according to the reaction scheme shown below in Fig. 1 A. The total concentration of DMN was kept at 3 mM. The concentration of steady state free Ca2+ was determined with a Ca2+-selective minielectrode (Györke et al. 1994). The local Ca2+ changes near the bilayer were calibrated by transforming the bilayer aperture into a Ca2+ electrode, using Ca2+ ionophore resin (Györke et al. 1994). The potential of the Ca2+ electrode was measured with 0.2 mV precision using the patch-clamp amplifier in current-clamp mode. The increase in free steady state Ca2+ after photolysis was plotted as a function of flash intensity and free Ca2+ before the flash to construct a calibration curve (see Fig. 1 B). The proportion of DMN photolyzed at a given free Ca2+ and flash intensity was calculated from the pre- and post-flash steady state free Ca2+, using parameters taken from the literature (Ellis-Davies et al. 1996; Escobar et al. 1997; Table ). The time course of Ca2+ concentration changes in a particular experiment was reconstructed from the above data, using the published set of differential equations and kinetic parameters of DM-nitrophen complexation and photolysis (Ellis-Davies et al. 1996; Escobar et al. 1997). Computations were performed with a program written in Mathematica (version 3.0; Wolfram Research).


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

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

Defining the properties of the rapid Ca2+ spikes. (A) The reaction scheme of DMN complexation and photolysis (Ellis-Davies et al. 1996). The light-induced transitions of DMN into the activated states are assumed to be irreversible and instantaneous, and only a fraction of DMN (ΔDMN; 0.5–2% in our experiments) undergoes this reaction step. Other transitions in the scheme are reversible. (B) The increase in steady state Ca2+ after the flash plotted as a function of preflash steady state Ca2+ in five independent experiments. The total DMN concentration was always 3 mM. Different symbols denote different power settings of the laser flash. For a given flash energy the log–log plot of ΔCa2+ vs. Ca2+ was linear and was used to precalibrate flash energy in a given experiment. (C) The time course of a typical calcium spike reconstructed from the pre- and postflash steady state Ca2+ concentrations. The on and off portions of the calcium profile could be fitted by monoexponential functions, giving τon = 6.5 ± 1.5 μs and τoff = 106 ± 1 μs.
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Related In: Results  -  Collection

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Figure 1: Defining the properties of the rapid Ca2+ spikes. (A) The reaction scheme of DMN complexation and photolysis (Ellis-Davies et al. 1996). The light-induced transitions of DMN into the activated states are assumed to be irreversible and instantaneous, and only a fraction of DMN (ΔDMN; 0.5–2% in our experiments) undergoes this reaction step. Other transitions in the scheme are reversible. (B) The increase in steady state Ca2+ after the flash plotted as a function of preflash steady state Ca2+ in five independent experiments. The total DMN concentration was always 3 mM. Different symbols denote different power settings of the laser flash. For a given flash energy the log–log plot of ΔCa2+ vs. Ca2+ was linear and was used to precalibrate flash energy in a given experiment. (C) The time course of a typical calcium spike reconstructed from the pre- and postflash steady state Ca2+ concentrations. The on and off portions of the calcium profile could be fitted by monoexponential functions, giving τon = 6.5 ± 1.5 μs and τoff = 106 ± 1 μs.
Mentions: Fast changes of the Ca2+ concentration in the microenvironment of the reconstituted channel were performed by flash photolysis of DM-nitrophen (Calbiochem Corp.) as described previously (Györke and Fill 1993; Györke et al. 1994). Intense, 9-ns long UV laser flashes produced by a pulsed, frequency-tripled, Nd:YAG laser (Spectra-Physics) were applied through a fused silica fiber optics (450 μm diameter) positioned perpendicular to the bilayer surface (100 μm diameter) so that the whole volume between the fiber optics and the bilayer was illuminated evenly and instantaneously. The amplitude and time course of Ca2+ after the flash were determined from the concentration of total and free DMN and Ca2+, and from the proportion of DMN photolyzed during the flash according to the reaction scheme shown below in Fig. 1 A. The total concentration of DMN was kept at 3 mM. The concentration of steady state free Ca2+ was determined with a Ca2+-selective minielectrode (Györke et al. 1994). The local Ca2+ changes near the bilayer were calibrated by transforming the bilayer aperture into a Ca2+ electrode, using Ca2+ ionophore resin (Györke et al. 1994). The potential of the Ca2+ electrode was measured with 0.2 mV precision using the patch-clamp amplifier in current-clamp mode. The increase in free steady state Ca2+ after photolysis was plotted as a function of flash intensity and free Ca2+ before the flash to construct a calibration curve (see Fig. 1 B). The proportion of DMN photolyzed at a given free Ca2+ and flash intensity was calculated from the pre- and post-flash steady state free Ca2+, using parameters taken from the literature (Ellis-Davies et al. 1996; Escobar et al. 1997; Table ). The time course of Ca2+ concentration changes in a particular experiment was reconstructed from the above data, using the published set of differential equations and kinetic parameters of DM-nitrophen complexation and photolysis (Ellis-Davies et al. 1996; Escobar et al. 1997). Computations were performed with a program written in Mathematica (version 3.0; Wolfram Research).

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