The broad objective of this research is to resolve molecular interactions that regulate the force and rate of the heartbeat. In cardiac muscle, as in skeletal muscle, contraction is activated by Ca2+ released from the sarcoplasmic reticulum (SR) via intracellular channels known as ryanodine receptors (RyRs). Proposed studies are focused on defining structural and functional interactions between RyR channel s and the regulatory protein calmodulin (CaM), which is postulated to act as a mobile, Ca2+-sensing channel subunit .
Aim 1 is to determine the mechanism by which CaM functions as an isoform-specific modulator of RyR Ca2+ sensitivity.
Aim 2 is to characterize CaM and FKBP binding sites on purified RyRs, and identify fluorescent derivatives that retain high-affinity RyR binding.
Aim 3 is to define distance relationships and Ca2+ dependent structural changes within the macromolecular RyR using fluorescent CaM and FKBP derivatives, in combination with fluorescent RyR fusion proteins, for measurements of fluorescence resonance energy transfer (FRET). Newly-identified point mutants of CaM that selectively abolish either stimulatory or inhibitory interactions with the RyRs will provide valuable tools for characterizing the modes of CaM-RyR binding and for resolving the mechanism through which CaM functions as a molecular switch modulating channel activity. Environment-sensitive, fluorescent CaM derivatives developed in preliminary studies will allow direct monitoring of changes in CaM Ca2+ affinity mediated via interactions with the intact, functional, RyR1, RyR2, and RyR3 isoforms. Structural data based on mutagenesis, site-directed labeling, and FRET will be supported by functional assays of channel activity, including single channel recordings, radioligand binding, and Ca2+ flux determinations. Together, available techniques and reagents will allow for the first time-resolved structural measurements within working RyR channels. Understanding the role of RyR regulatory proteins is a major challenge driving current research into the mechanisms that control SR Ca2+ release during excitation-contraction (EC) coupling, and CaM, in particular, evokes pronounced and defined changes in these channels' response to Ca2+. Proposed studies will provide fundamental structural and mechanistic insights into channel regulation, and thereby aid in the search for improved strategies targeting defective SR Ca2+ handling in arrhythmias and heart failure. ? ?
