PHOSPHORYLATION MODULATES THE FUNCTION OF THE CALCIUM-RELEASE CHANNELOF SARCOPLASMIC-RETICULUM FROM CARDIAC-MUSCLE

The cardiac calcium release channel (CRC) of sarcoplasmic reticulum ve sicles was incorporated into planar lipid membranes to evaluate modula tion of channel activity by phosphorylation and dephosphorylation. For this purpose a microsyringe application directly to the membrane was used to achieve sequential and multiple treatments of channels with hi ghly purified kinases and phosphatases. Cyclic application of protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CalPK) and potato acid phosphatase or protein phosphatase 1 revealed a channe l block by Mg2+ (similar to mM), that is referable to dephosphorylated states of the channel, and that the Mg2+ block could be removed by ph osphorylation of the CRC by either PKA or CalPK, By contrast, activati on of endogenous CalPK (end CalPK) led to channel closure which could be reversed by dephosphorylation using potato acid phosphatase or prot ein phosphatase 1, Calmodulin by itself (which activates end CalPK in the presence of MgATP) blocks the channel in the dephosphorylated stat e, which can be overcome by treatment with CalPK but not PKA. Our find ings reveal important insights regarding channel regulation of the rya nodine receptor: 1) the calcium release channel must be phosphorylated to be in the active state at conditions approximating physiological M g2+ concentrations (similar to mM); and 2) there are multiple sites of phosphorylation on the calcium release channel with different functio nal consequences, which may be relevant to the regulation of E-C coupl ing, Phosphorylation of the CRC may be involved in recruitment of acti ve channels, and/or it may be directly involved in each Ca2+ contracti on cycle of the heart, For example, Ca2+ release may require phosphory lation of the CRC by protein kinases at sites which overcome the block by Mg2+, Inactivation may involve CRC block by calmodulin and/or phos phorylation by endogenous CalPK at the junctional face membrane.