MODULATION OF CALCIUM FLUXES IN HEART AND SMOOTH MUSCLE

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The long term goal of our ongoing program is to understand excitation- contraction (E-C) coupling in muscle (skeletal, heart and smooth muscle). The emphasis is on the structure, function and regulation of the intracellular calcium release channels (ICRCs), a new class of channels characterized by their large size and four-fold symmetry. ICRCs are important in mobilizing Ca2+ from intracellular stores for cell signalling. There are two types, the ryanodine receptor (RyR) and the IP3 receptor (IP3R). Two recent findings in our laboratory provide the basis of key proposed studies regarding the modulation of the RyR receptor. 1)Phosphorylation of terminal Cisternae of sacroplasmic reticulum by protein kinases renders the channel active, whereas phosphatase action renders the channel inactive, in the presence of approximately physiological [Mg2+].; 2) FK binding protein (FKBP), the receptor for the immunosuppressive drug FK5O61 is tightly bound to the ryanodine receptor isoforms of skeletal muscle (RyR-1) and heart (RyR-2). There are 7 aims: 1) Characterize the nature of the modulation of RyR-1 and RyR-2 activity by protein kinases and phosphatases 2) Study the modulation of the IP3 R from smooth muscle by phosphorylation/dephosphorylation with protei kinases/phosphatases. The experimental approach is similar to that for the RyR. (See Aim 1). 3) Determine surface topology of IP3 R from smooth muscle by proteolysis sensitivity mapping. That is, the intact receptor will be cleaved by selective proteolytic digestion and the peptides formed identified by chemical sequencing. Each peptide identifies a specific peptide bond on the surface of the receptor. 4) Continue three-dimensional structure analysis studies of the RyR from heart and skeletal muscle and initiate studies with the IP3 R (together with Dr. Terrence Wagenknecht and colleagues). 5) Assess the physiological relevance and functional consequence of the association of the FKBP with skeletal muscle and heart ryanodine receptors. 6) Define the surface interfaces on FKBP and on the RyR receptor in the association of specific FKBP isoforms with the RyR for both skeletal muscle (FKBP12/RyR1) and heart (FKBP-C/RyR2) 7) Evaluate the role of FKBP and phosphorylation/dephosphorylation in modulating E-C coupling in skeletal muscle and heart. Our program is designed to correlate structure/function relationships of the ICRCs and their physiological relevance to calcium homeostasis and E-C coupling in muscle.