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

The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a m icrosyringe applicator to achieve sequential and multiple treatments w ith highly purified kinases and phosphatases applied directly at the b ilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bil ayers, and single-channel currents were measured at zero holding poten tial, at 0.15 mu M free Ca2+, +/-0.5 mM ATP and +/-2.6 mM free Mg2+. S equential dephosphorylation and rephosphorylation rendered the CRC sen sitive and insensitive to block by Mg2+, respectively. Channel recover y from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK ii). Somewhat di fferent characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was re stored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found t o be ''dephosphorylated'' (inactive) in 60%, and ''phosphorylated'' (a ctive) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconv ertable by treatment with exogenously added protein kinases and phosph atases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK ) had an opposite action to exogenous CalPK II. Previously, dephosphor ylated channels using PPT (Mg2+ absent) were blocked in the closed sta te by action of endogenous CalPK. This block was removed to normal act ivity by the action of either PPT or by exogenous CalPK II. Our findin gs are consistent with a physiological role for phosphorylation/dephos phorylation in the modulation of the calcium release channel of sarcop lasmic reticulum from skeletal muscle. A corollary of our studies is t hat only the phosphorylated channel is active under physiological cond itions (mM Mg2+). Our studies suggest that phosphorylation can be at m ore than one site and, depending on the site, can have different funct ional consequences on the CRC.