RIS ID
120849
Abstract
A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α1S subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.
Publication Details
Tae, H., Cui, Y., Karunasekara, Y., Board, P. G., Dulhunty, A. F. & Casarotto, M. G. (2011). Cyclization of the intrinsically disordered a1Sdihydropyridine receptor II-III loop enhances secondary structure and in vitro function. Journal of Biological Chemistry, 286 (25), 22589-22599.