The long-term objective of this project is to identify the molecular interactions underlying skeletal-type excitation- contraction (EC) coupling, the process which links electrical excitation to muscular contraction. EC coupling hinges upon a functional interaction between the ryanodine receptor (RyR), a Ca2+-release channel located in the sarcoplasmic reticulum (SR), and the dihydropyridine receptor (DHPR), a voltage-gated Ca2+ channel which is located in the plasma membrane and contains alpha1S as its principal subunit. A number of experimental approaches will be used to probe the interaction between the DHPR and RyR, including the use of patch clamping, Ca2+ indicator dyes, electron microscopy and molecular biology. The proposal's first specific aim is to establish the determinants that cause DHPRs to target to junctions between the plasma membrane and sarcoplasmic reticulum. This will be accomplished by expression in dysgenic (alpha1S-null) myotubes of green fluorescent protein (GFP) tagged chimeras of alpha1S and alpha1H (a distantly related Ca2+ channel), and by a yeast two-hybrid screen of a muscle library using a potential targeting domains of alpha1S as baits.
The second aim i s to use alpha1S/alpha1H chimeras expressed in dysgenic myotubes as a means of testing whether the beta subunit of the DHPR is required for skeletal-type coupling, to determine whether the primary sequence of cytoplasmic domains outside the II-III loop are critical for coupling, and to identify the sequence(s) of alpha1S that cause DHPRs to be organized into """"""""tetrads"""""""".
The third aim i s to test whether EC coupling depends upon conformational changes of the alpha1S II-III loop, which will be examined by means of introducing structural perturbations into the regions of the loop which surround the """"""""critical domain"""""""" of the loop. One perturbation to be tested is the introduction of the biotin acceptor domain, which specifies the metabolic addition of biotin to a small number of native enzymes containing this essential cofactor.
The fourth aim i s to attempt to reconstitute skeletal-type coupling by expressing a minimal set of muscle proteins in a non-muscle cell.
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