The central paradox of cardiac excitation-contraction (E-C) coupling is that Ca2+-induced Ca2+ release (CICR), an inherently self-regenerating process, is finely graded by surface membrane Ca2+ currents. Recently, I showed that the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors, RyRs) from cardiac muscle exhibit the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors, RyRs) from cardiac muscle exhibit a unique adaptive behavior, characterized by the ability of individual channels RyRs to respond to incremental increases in Ca2+ by transient bursts of activity. This phenomenon could account for the graded nature of CICR in vivo and may be a fundamental property of all intracellular Ca2+ release channels, including the inositol triphosphate receptors (IP3R).
The aim of this proposal is to determine the molecular basis of this phenomenon and define its role in controlling Ca2+ release in vivo. Measurements of single channel activity and Ca2+ release from SR microsomes in response to step [Ca2+] changes, induced by photolysis of caged-Ca2+, will be performed to define the dynamic properties of the RyR/Ca2+ channels (e.g., rate of adaptation, nonstationary Ca2+-dependence) in the presence of physiologically relevant ligands (e.g., ATP, Mg2+, Ca2+). Defining these """"""""elementary"""""""" properties of Ca2+ release is essential for discriminating between alternative hypotheses of how CICR is regulated in vivo. For example, an adaptation with sufficiently rapid kinetics could operate by countering the intrinsic positive feedback of CICR. A slow shift in RyR Ca2+ sensitivity could provide a basis for a different mechanism, which operates by fine-tuning the Ca2+ sensitivity of the release to maintain a stable graded CICR. To determine how single channel adaptation is expressed in situ, experiments involving measurements of surface membrane Ca2+ current, intracellular [Ca2+] and photolysis of caged-Ca2+ will be performed in patch-clamped isolated cardiac myocytes. To define the mechanism of adaptation, specific hypotheses for molecular mechanisms (i.e., cis-trans isomerization, phosphorylation/dephosphorylation, etc._ will be tested experimentally in single channel and macroscopic measurements. Understanding how Ca2+ release is regulated is important since it represents a strategic site for therapeutic intervention. Further, defining the mechanism of regulation of cardiac Ca2+ release channels has broader significance since the regulation of analogous channels (IP3Rs and RyRs) in other tissues are not well understood.
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