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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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Response of single RyR channels to a sequence of two identical laser flashes. (A, top) The time course of the Ca2+ stimuli estimated by the calcium cup electrode (dashed line) and calculated from the pre- and postflash steady state Ca2+ concentrations (full line). (Middle) Representative single-channel records measured at +40 mV. The flashes were applied at t = 60 and 230 ms (dotted lines). Only the first 400 ms of 3.2-s-long traces are shown. (Bottom) Ensemble current constructed from 54 episodes. The vertical calibration denotes 20 and 5 pA for the center and bottom, respectively. (B) The time course of the ensemble current at a compressed time scale. Open probability in response to the second laser flash decayed monoexponentially with a time constant of 880 ms (white line).
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Figure 3: Response of single RyR channels to a sequence of two identical laser flashes. (A, top) The time course of the Ca2+ stimuli estimated by the calcium cup electrode (dashed line) and calculated from the pre- and postflash steady state Ca2+ concentrations (full line). (Middle) Representative single-channel records measured at +40 mV. The flashes were applied at t = 60 and 230 ms (dotted lines). Only the first 400 ms of 3.2-s-long traces are shown. (Bottom) Ensemble current constructed from 54 episodes. The vertical calibration denotes 20 and 5 pA for the center and bottom, respectively. (B) The time course of the ensemble current at a compressed time scale. Open probability in response to the second laser flash decayed monoexponentially with a time constant of 880 ms (white line).

Mentions: Previous studies of RyR activation by photolysis of DMN (Györke and Fill 1993, Györke and Fill 1994; Valdivia et al. 1995) showed only sustained RyR responses decaying (i.e., adapting) with a time constant of ∼1 s and displayed no brief responses demonstrated in the present study. To explore the relationship between the rapid and sustained responses, we performed measurements of RyR activity during two sequential laser flashes of equal intensity (Fig. 3 A, top). It can be seen that while the first flash elicited predominantly single openings (Ca2+ spike response), the second pulse triggered mostly multiple openings (adaptation response). The corresponding changes in free [Ca2+] (continuous line) calculated from the Ca2+ electrode response (dashed line) using published parameters of complexation and photolysis of DMN (Ellis-Davies et al. 1996) are also presented (Fig. 3 A). The first flash elicited a Ca2+ spike followed by a small steady [Ca2+] elevation; the second flash elicited a similar Ca2+ spike, which was followed by a steady [Ca2+] elevation to a significantly higher level. The increase in steady component of the Ca2+ signal during successive flashes is due to a gradual increase in the saturation of DMN by Ca2+, leaving less DMN for rebinding of Ca2+ after the flash. These results clearly show that the adaptation behavior is determined by the steady component of the [Ca2+] signal.


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

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

Response of single RyR channels to a sequence of two identical laser flashes. (A, top) The time course of the Ca2+ stimuli estimated by the calcium cup electrode (dashed line) and calculated from the pre- and postflash steady state Ca2+ concentrations (full line). (Middle) Representative single-channel records measured at +40 mV. The flashes were applied at t = 60 and 230 ms (dotted lines). Only the first 400 ms of 3.2-s-long traces are shown. (Bottom) Ensemble current constructed from 54 episodes. The vertical calibration denotes 20 and 5 pA for the center and bottom, respectively. (B) The time course of the ensemble current at a compressed time scale. Open probability in response to the second laser flash decayed monoexponentially with a time constant of 880 ms (white line).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230654&req=5

Figure 3: Response of single RyR channels to a sequence of two identical laser flashes. (A, top) The time course of the Ca2+ stimuli estimated by the calcium cup electrode (dashed line) and calculated from the pre- and postflash steady state Ca2+ concentrations (full line). (Middle) Representative single-channel records measured at +40 mV. The flashes were applied at t = 60 and 230 ms (dotted lines). Only the first 400 ms of 3.2-s-long traces are shown. (Bottom) Ensemble current constructed from 54 episodes. The vertical calibration denotes 20 and 5 pA for the center and bottom, respectively. (B) The time course of the ensemble current at a compressed time scale. Open probability in response to the second laser flash decayed monoexponentially with a time constant of 880 ms (white line).
Mentions: Previous studies of RyR activation by photolysis of DMN (Györke and Fill 1993, Györke and Fill 1994; Valdivia et al. 1995) showed only sustained RyR responses decaying (i.e., adapting) with a time constant of ∼1 s and displayed no brief responses demonstrated in the present study. To explore the relationship between the rapid and sustained responses, we performed measurements of RyR activity during two sequential laser flashes of equal intensity (Fig. 3 A, top). It can be seen that while the first flash elicited predominantly single openings (Ca2+ spike response), the second pulse triggered mostly multiple openings (adaptation response). The corresponding changes in free [Ca2+] (continuous line) calculated from the Ca2+ electrode response (dashed line) using published parameters of complexation and photolysis of DMN (Ellis-Davies et al. 1996) are also presented (Fig. 3 A). The first flash elicited a Ca2+ spike followed by a small steady [Ca2+] elevation; the second flash elicited a similar Ca2+ spike, which was followed by a steady [Ca2+] elevation to a significantly higher level. The increase in steady component of the Ca2+ signal during successive flashes is due to a gradual increase in the saturation of DMN by Ca2+, leaving less DMN for rebinding of Ca2+ after the flash. These results clearly show that the adaptation behavior is determined by the steady component of the [Ca2+] signal.

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