Expression and functional characterization of the cardiac muscle ryanodine receptor Ca2+ release channel in Chinese hamster ovary cells

To study the function and regulation of the cardiac ryanodine receptor (RyR2) Ca2+ release channel, we expressed the RyR2 proteins in a Chinese hamster ovary (CHO) cell line, and assayed its function by single channel current recording and confocal imaging of intracellular Ca2+ ([Ca2+](i)). The 16-kb cDNA encoding the full-length RyR2 was introduced into CHO cells using lipofectAmine and electroporation methods. Incorporation of microsomal membrane vesicles isolated from these transfected cells into lipid bilayer membrane resulted in single Ca2+ release channel activities similar to those of the native Ca2+ release channels from rabbit cardiac muscle SR membranes, both in terms of gating kinetics, conductance, and ryanodine modification. The expressed RyR2 channels were found to exhibit more frequent transitions to subconductance states than the native RyR2 channels and RyR1 expressed in CHO cells. Caffeine, an exogenous activator of RyR, induced release of [Ca2+](i) from these cells. Confocal imaging;of cells expressing RyR2 did not detect spontaneous or caffeine-induced local Ca2+ release events (i.e., "Ca2+ sparks") typically seen in cardiac muscle. Our data show that the RyR2 expressed in CHO cells forms functional Ca2+ release channels. Furthermore, the lack of localized Ca2+ release events in these cells suggests that Ca2+ sparks observed in cardiac muscle may involve cooperative gating of a group of Ca2+ release channels and/or their interaction with muscle-specific proteins.