Patients with myasthenia gravis can have antibodies against skeletal muscle ryanodine receptor (Ry1), the sarcoplasmic reticulum calcium-release channel, which plays a crucial role in excitation-contraction coupling. We have screened a panel of overlapping Ry1 fusion proteins with Ry1 antibody-containing myasthenia gravis sera to identify the main immunogenic region. The pc2 Ry1 fusion protein representing a Ry1 region close to the N-terminus (residues 799-1172) was identified as the main immunogenic region for the antibodies. The binding kinetics of the Ry1 antibodies to the pc2 Ry1 fusion protein were tested using an optical biosensor. Ry1 antibodies in the IgG fraction from sera of patients with myasthenia gravis bound with high affinity and with a stoichiometry of 1:1. The functional effect of these Ry1 antibodies was tested in an in vitro Ca2+-release assay. The Ry1 antibodies induced a twofold increase of the half-maximal concentration for 4-Cl-m-cresol-induced Ca2+ release from terminal cisternae vesicles but had no effect on V(max). The effect on 4-Cl-m-cresol-induced Ca2+ release was specific, as preincubation of the active IgG fraction with the pc2 Ry1 fusion protein abolished the inhibition. These data suggest that the Ry1 sequence defined by residues 799-1172 is involved in the regulation of Ry1 function, and that this regulation could be functionally affected in vivo in patients with myasthenia gravis.