ROLE OF MALIGNANT HYPERTHERMIA DOMAIN IN THE REGULATION OF CA2-MUSCLESARCOPLASMIC-RETICULUM( RELEASE CHANNEL (RYANODINE RECEPTOR) OF SKELETAL)

A fusion protein encompassing Gly(341) of the skeletal muscle ryanodin e receptor was used to raise monoclonal antibodies; epitope mapping de monstrates that monoclonal antibody 419 (mAb419) reacts with a sequenc e a few residues upstream from Gly(341). The mAb419 was then used to p robe ryanodine receptor (EYE) functions. Our results show that upon in cubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [H-3]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-max imal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 v ersus 1.2 mu M). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbatio n by the Ab of the intramolecular interaction between the immunopositi ve region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIA-core (Pharmacia Biotec h Inc.): we show that the immunopositive EYE polypeptide is able to in teract with the native RYR complex. Ligand overlays with immunopositiv e digoxigenin-RYR fusion protein indicate that such an interaction mig ht occur with a calmodulin binding domain (defined by residues 3010-32 25) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR cha nnel activity is due to the perturbation of an intramolecular interact ion between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.