The ryanodine receptor (RyR)/calcium (Ca) release channel is required for excitation- contraction (EC) coupling in cardiac and skeletal muscle. Although the primary structures of the RyR/Ca release channels have been determined from cDNA cloning, most of the functional domains of the channels have not yet been identified. Therefore, how channel properties are determined by the channel structure is not well understood. The goal of this proposal is to use the cloned expressed RyR to increase our knowledge of how fundamental channel properties are related to structure.
The aims of this proposal are made feasible by the development of a model system for heterologous functional expression of RyR in insect cells and by the identification of an endogenous channel associated protein, FKBP, that modulates channel gating specifically by: 1) increasing the frequency of the full conductance state of the channel; 2) increasing mean open time (t); and 3) decreasing open probability (Po) after caffeine activation.
Aim 1 proposes to identify the FKBP binding site on skeletal RyR1.
Aim 2 proposes to characterize the modulation of cardiac RyR2 gating by a novel form of FKBP.
Aim 3 proposes to determine whether the proline isomerase activity of FKBP is required for channel modulation. RyR molecules containing mutations in FKBP binding domains will be expressed in muscle cells that lack the RyR gene to determine the physiologic role of FKBP in the RyR channel complex. Elucidation of the structure/function relationships of RyR will help us understand the regulation of Ca release from sarcoplasmic reticulum.
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