| The sarcoplasmic reticulum (SR) calcium release channel, also known as the ryanodine receptor (RyR), is an essential component of excitation-contraction (E-C) coupling in striated muscle cells. Mammalian skeletal (RyR1) and cardiac (RyR2) isoforms of RyR share a high degree of similarity, but differ significantly in three regions of divergence referred to as D1, D2 and D3. An important difference between skeletal and cardiac muscle involves RyR regulation by cytosolic free Ca2+. Free cytosolic [Ca2+] regulates RyR function through interactions with high- and low-affinity Ca2+ binding sites. The high-affinity site is believed to bind C a2+ at micromolar concentrations resulting in opening of the channel and release of Ca2+ from the SR. The low-affinity site is thought to bind Ca2+ at millimolar concentrations and to result in inhibition of Ca2+ release. Cardiac RyR is more resistant to inhibition by millimolar Ca2+ than its skeletal counterpart. The divergent region D3 is highly negatively charged in RyR1 compared to RyR2 and has been hypothesized to form a low-affinity Ca2+ binding site.; To understand the role of the D3 region in Ca2+-dependent regulation of the Ca2+ release channel, we generated mutant constructs of RyR1 and RyR2 rabbit cDNA and expressed the respective proteins in Chinese hamster ovary (CHO) cells that normally do not express RyR. The functional effects of these mutations were studied using, fluorescent measurement of RyR-mediated intracellular Ca2+ release, lipid bilayer reconstitution of single Ca2+ release channels as well as [3H]ryanodine binding assays. Deletion of the D3 region from RyR1 resulted in functional Ca2+ release channels that were resistant to Ca 2+-dependent inactivation when compared to full-length RyR1, but that also activated at lower [Ca2+]. Deletion of a large segment of RyR2 that included the D3 region resulted in channels that did not appear to inactivate in response to millimolar [Ca2+]. The inactivation function, however, could be restored by expression of skeletal D3 in a chimeric construct involving the cardiac deletion mutant. [3H]Ryanodine binding studies revealed that the effect of D3 was modulatory to Ca 2+-dependent regulation of RyR through close interaction with Ca 2+ activation and inactivation domains of the Ca2+ release channel. |
