Key result
Mutation of three critical SPRY2 F loop residues abolished in vitro binding to the ASI/basic region but did not alter Ca2+ release during ligand- and depolarization-induced RyR1 activation in myotubes.
The SPRY2 F loop interacts with the ASI/basic region to regulate RyR1 channel activity in vitro, but this interaction does not influence skeletal muscle excitation-contraction coupling in intact cells.
SPRY2-ASI interaction is dispensable for RyR1 Ca2+ release in myotubes; leaves open alternative mechanisms for skeletal EC coupling.
The second of three SPRY domains (SPRY2, S1085 -V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.
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Tae et al. (2011) studied None (Basic Science). SPRY2 F loop mutations (E1108A, D1112A, E1114A) vs. Wild-type RyR1 was evaluated on Ca2+ release during ligand- and depolarization-induced RyR1 activation. Mutation of three critical SPRY2 F loop residues abolished in vitro binding to the ASI/basic region but did not alter Ca2+ release during ligand- and depolarization-induced RyR1 activation in myotubes.
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