The cardiac calcium release channel (CRC) of sarcoplasmic reticulum vesicles was incorporated into planar lipid membranes to evaluate modulation 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 highly 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 channel block by Mg2+ (-mM), that is referable to dephosphorylated states of the channel, and that the Mg2+ block could be removed by phosphorylation of the CRC by either PKA or CalPK. By contrast, activation of endogenous CalPK (end CalPK) led to channel closure which could be reversed by dephosphorylation using potato acid phosphatase or protein phosphatase 1. Calmodulin by itself (which activates end CalPK in the presence of MgATP) blocks the channel in the dephosphorylated state, which can be overcome by treatment with CalPK but not PKA. Our findings reveal important insights regarding channel regulation of the ryanodine receptor: 1) the calcium release channel must be phosphorylated to be in the active state at conditions approximating physiological Mg2+ concentrations (-mM); and 2) there are multiple sites of phosphorylation on the calcium release channel with different functional consequences, which may be relevant to the regulation of E-C coupling. Phosphorylation of the CRC may be involved in recruitment of active channels, and/or it may be directly involved in each Ca2+ contraction cycle of the heart. For example, Ca2+ release may require phosphorylation of the CRC by protein kinases at sites which overcome the block by Mg2+. Inactivation may involve CRC block by calmodulin and/or phosphorylation by endogenous CalPK at the junctional face membrane.

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The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.

Inositol polyphosphate receptor and clathrin assembly protein AP-2 are related proteins that form potassium-selective ion channels in planar lipid bilayers (receptor-mediated Clathrin-coated vesicles similar to fraction D described by Keen et al. (11) were prepared from three or four whole bovine brains.Clathrin and AP-2 were partially purified from the coated vesicles essentially as described by Keen et al. (11). Briefly, coated vesicles (1.5 g of protein) were extracted with 0.5 M Tris Cl (200 ml) in the absence of detergents. The extract was precipitated in 50% ammonium sulfate, resuspended in 15-20 ml of buffer B (0.5 M Tris Cl, pH 7.0/50 mM NaMes/0.5 mM EGTA/0.25 mM MgCl2/10% glycerol), applied to an HPLC gel exclusion column (2.1 x 60 cm TSK G-4000SW or TSK G-3000 SW column; TosoHaas, Philadelphia) equilibrated in buffer B, and eluted at 2.5 ml/min, collecting 3-ml fractions. Fractions from the G-4000 column enriched in clathrin (fractions 33-40) were pooled, dialyzed overnight against buffer C (50 mM Tris Cl, pH 8.3/1 MM EDTA/1 mM dithiothreitol/ 10% glycerol), concentrated to about 1 ml by ultrafiltration (Centriprep 30; Amicon), frozen in liquid nitrogen, and stored at -80TC. Fractions from the G-3000 or G4000 column enriched in AP-2 (typically fractions 42-49 of the G-4000 column) were pooled and dialyzed overnight against 2-4 liters of buffer C. In some preparations, the dialysate was concentrated to about 1 ml by ultrafiltration; we refer to this 8976The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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