The cardiac ryanodine receptor calcium-release channel (RyR2) and the smooth-muscle large-conductance calcium-and-voltage-activated potassium (BK) channel are vital participants in normal and pathological cardiovascular physiology. Each is a tetramer of pore-forming subunits in a complex with auxiliary subunits that allosterically modulate channel behavior. FK506-binding protein (FKBP12.6) modulates RyR2, and betal modulates BK channel alpha subu'nit. In addition, these channels are allosterically modulated by ligands with therapeutic potential, such as RyR2 by JTV519. To understand the mechanisms of these allosteric interactions, the amino acid residues in the binding sites for FKBP12.6 and for JTV519 on RyR2 and for betal subunit on BK channel alpha subunit will be identified. Furthermore, changes in the contacts in different functional states of the channels will be determined. Binding-site residues in RyR2 in cardiac sarcoplasmic reticulum membrane, and in RyR2 and BK channel heterologously expressed in cultured cells, will be tagged by photoaffinity labeling, chemical crbsslinking, and foot-printing methods. The residues will be identified by protein cleavage, isolation of labeled peptides, mass spectrometry and micro-sequencing. In addition, cross-linking reactions will be directed to cysteine residues substituted by site-directed mutagenesis at specific locations on the target proteins, and cross-linking will be detected by Western blotting. Modifications of known methods are proposed to make feasible the detailed characterization of binding sites on a low abundance channel like BK and on a very large protein like the RyR2 subunit. The results will provide insights into the molecular mechanisms of allosteric interactions in these two channels and will provide molecular structural bases for cardiovascular therapeutics targeted at these channels.
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