Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum.
A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.
Zorzato
F
F
Institute of General Pathology, University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy.
Menegazzi
P
P
Treves
S
S
Ronjat
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000276:immunology / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine; D009124:Muscle Proteins / Q000276:immunology / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium; D005998:Glycine