The elusive role of the SPRY2 domain in RyR1.
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.
Howard Florey Institute, Parkville, Victoria, Australia.
HanShen
H
Tae
Lan
L
Wei
Hermia
H
Willemse
Shamaruh
S
Mirza
Esther M
EM
Gallant
Philip G
PG
Board
Robert T
RT
Dirksen
Marco Giovani
MG
Casarotto
Angela
A
Dulhunty
D017398:Alternative Splicing; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D004093:Dihydropteridine Reductase / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D007700:Kinetics; D008565:Membrane Proteins; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D005722:gamma-Glutamylcyclotransferase / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C485561:SPRY2 protein, human; D004093:Dihydropteridine Reductase; D005722:gamma-Glutamylcyclotransferase
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