Ryanodine receptor type III (Ry3R) identification in mouse parotid acini. Properties and modulation of [3H]ryanodine-binding sites.
Immunoblot analysis and [3H]ryanodine binding were used to characterize and identify ryanodine receptors (RyRs) in nonexcitable mouse parotid acini. Western analysis revealed ryanodine receptor type III (Ry3R) to be the only detectable isoform in parotid microsomal membranes. Binding of [3H]ryanodine to microsomal fractions was dependent on Ca2+, salt, pH, and temperature. At 23 degrees C, and in the presence of 0.5 M KCl and 100 microM Ca2+, [3H]ryanodine bound specifically to membranes with high affinity (Kd = 6 nM); maximum binding capacity (Bmax) was 275 fmol/mg protein. Mg2+ and ruthenium red inhibited [3H]ryanodine binding (IC50 = 1.4 mM and 0.5 microM, respectively). 4-Chloro-3-ethylphenol enhanced the binding of [3H]ryanodine 2.5-fold; whereas ATP and caffeine were much less efficacious toward activating Ry3R (56% and 18% maximal enhancement, respectively). Bastadin, a novel modulator of the 12-kDa FK506 binding protein.RyR complex, increased [3H]ryanodine binding 3-4-fold by enhancing Kd. The immunosuppressant FK506 enhanced [3H]ryanodine receptor occupancy at >100 microM and antagonized the action of bastadin, suggesting that an immunophilin modulates Ry3R in parotid acini. These results suggest that Ry3R may play an important role in Ca2+ homeostasis in mouse parotid acini.
DiJulio
D H
DH
Department of Oral Biology, University of Washington, Seattle, Washington 98195, USA.
Watson
E L
EL
Pessah
I N
IN
Jacobson
K L
KL
Ott
S M
SM
Buck
E D
ED
Singh
J C
JC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D010306:Parotid Gland / Q000737:chemistry / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel
D015220:Calcium Channels; D002148:Calmodulin-Binding Proteins; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D015636:Magnesium Chloride; D012433:Ryanodine; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002110:Caffeine; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate