Synthetic localized calcium transients directly probe signalling mechanisms in skeletal muscle

Abstract

Key points• The signal for skeletal muscle contraction is a rapid increase in cytosolic Ca 2+ concentration, which requires the coordinated opening of ryanodine receptor (RyR) channels in the sarcoplasmic reticulum.• Channel opening is controlled by voltage-sensing dihydropyridine receptors (DHPRs) of plasma membrane and T tubules. Whether or not their signal is amplified by Ca 2+ -induced Ca 2+ release (CICR) is controversial.• We used two-photon lysis of an advanced Ca 2+ cage to produce local Ca 2+ concentration transients that were large, fast, reproducible and quantifiable, while monitoring the cellular response with a dual confocal laser scanner.• Single frog muscle cells in physiological solutions responded to transients greater than 0.28 μM with propagated CICR waves.• Mouse cells did not respond to stimuli up to 8 μM, unless channel opening drugs were present.• We conclude that CICR contributes to physiological Ca 2+ release in frog but not mouse muscle.• Mice and presumably other mammals do have a capability for CICR that is normally inhibited.It could be manifested under special circumstances, including diseases.Abstract The contribution of Ca 2+ -induced Ca 2+ release (CICR) to trigger muscle contraction is controversial. It was studied on isolated muscle fibres using synthetic localized increases in Ca 2+ concentration, SLICs, generated by two-photon photorelease from nitrodibenzofuran (NDBF)-EGTA just outside the permeabilized plasma membrane. SLICs provided a way to increase cytosolic [Ca 2+ ] rapidly and reversibly, up to 8 μM, levels similar to those reached during physiological activity. They improve over previous paradigms in rate of rise, locality and reproducibility. Use of NDBF-EGTA allowed for the separate modification of resting [Ca 2+ ], trigger [Ca 2+ ] and resting [Mg 2+ ]. In frog muscle, SLICs elicited propagated responses that had the characteristics of CICR. The threshold [Ca 2+ ] for triggering a response was 0.5 μM or less. As this value is much lower than concentrations prevailing near channels during normal activity, the result supports participation of CICR in the physiological control of contraction in amphibian muscle. As SLICs were applied outside cells, the primary stimulus was Ca 2+ , rather than the radiation or subproducts of photorelease. Therefore the responses qualify as 'classic' CICR. By contrast, mouse muscle fibres did not respond unless channel-opening drugs were present at substantial concentrations, an observation contrary to the physiological involvement of CICR in mammalian excitation-contraction coupling. In mouse muscle, the propagating wave had a substantially lower release flux, which together with a much higher threshold justified the absence of response when drugs were not present. The differences in flux and threshold may be ascribed to the absence of ryanodine receptor 3 (RyR3) isoforms in adult mammalian muscle.

Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP

“…During excitation (and upon t-tubular depolarization), a conformational change in the DHPRs located in the t-tubule triggers the activation of the RyR1s present in terminal cisternae (53). These characteristics of coupled RyR1s may explain, at least in part, the recent observation that RyR1s under DHPR control do not seem to be activated by local CICR (17). Although electron microscopy studies showed that DHPRs may not physically communicate with all RyR1s (24,26), our results and previous reports (39) indicate that RyR1s are coupled through physical interactions.…”

Phosphatidylinositol 3-kinase inhibition restores Ca 2+ release defects and prolongs survival in myotubularin-deficient mice

“…21). In mammalian skeletal muscle, EC coupling requires direct interactions between t-tubule CAV1.1 and SR RYR1 channels, and Ca 2+ -induced Ca 2+ release is not physiologically involved under normal conditions (22). Proper t-tubule SR organization is thus compulsory for normal EC coupling function, and t-tubule disruption should annihilate the capacity of disconnected RYR1 channels to respond to t-tubule depolarization.…”

Waveless mammalian muscle

“…Further speculation could go as far as prospecting that, thus far, collected data tending to deny CICR function in mammalian fibres have missed fulfilling an anonymous crucial criterion for CICR to operate. At the moment, however, as suggested by Figueroa et al the most likely explanation for the difference is that RyR3 is the required component.…”

“…The paper by Figueroa et al in a recent issue of The Journal of Physiology further tackles the problem by exploring the archetypal issue of whether a pure rise in Ca 2+ can trigger physiologically significant Ca 2+ release in adult muscle fibres.…”