Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor

A negatively charged region of the N-terminal portion of the skeletal ryano dine receptor (RyR), located between residues 1872-1923, is involved in Ca2 +-dependent regulation of the Ca2+-release channel. This region is divergen t between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, a nd is known as D3. Ca2+ exerts important regulatory functions on the RyR, b eing involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca2+ concentration s respectively. To characterize the role of D3 in the Ca2+-dependent regula tion of the Ca2+-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (Delta D3-RyR1) using a heterologous expr ession system, [H-3]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca2+-depe ndent regulation of RyR1, but did not alter [H-3]ryanodine binding or the e ffect of other modulators on the RyR. Compared with full-length RyR1 (wt-Ry R1), the Ca2+ dependence curve of Delta D3-RyR1 is broader, reflecting incr eased sensitivity to Ca2+ activation and decreased sensitivity to Ca2+ inac tivation. In addition, Delta D3-RyR1 was more resistant to inhibition by Mg 2+. Comparison of the effect of caffeine on wt-RyR1 and Delta D3-RyR1 sugge sted that D3 is an important region of RyR that participates in Ca2+-depend ent activation and inactivation of the Ca2+-release channel.