Netticadan T, Xu A, Narayanan N
Department of Physiology, University of Western Ontario, London, Canada.
Arch Biochem Biophys. 1996 Sep 15;333(2):368-76. doi: 10.1006/abbi.1996.0403.
In cardiac muscle, a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the sarcoplasmic reticulum (SR) is known to phosphorylate the membrane proteins phospholamban, Ca(2+)-ATPase, and Ca(2+)-release channel (ryanodine receptor). Phosphorylation of phospholamban and Ca(2+)-ATPase is recognized to stimulate Ca2+ sequestration by the SR but the functional consequence of Ca2+ channel phosphorylation has not been clearly established. In this study, we investigated the effects of the SR Ca(2+)-release inhibitor, ruthenium red (RR), and the SR Ca(2+)-release activator, ryanodine (at submicromolar concentrations), on CaM kinase-mediated phosphorylation of the Ca(2+)-cycling proteins in rabbit cardiac SR. Incubation of SR with RR (5-30 microM) for 3 min at 37 degrees C resulted in marked (up to 85%) inhibition of Ca2+ channel phosphorylation (50% inhibition with 15 +/- 2 microM RR) by the endogenous membrane-associated CaM kinase. Phosphorylation of the Ca2+ channel by exogenously added multifunctional alpha CaM kinase II was also inhibited similarly by RR. Phosphorylation of the Ca(2+)-ATPase by endogenous and exogenous CaM kinase was inhibited only modestly (25-30%) by RR, and phospholamban phosphorylation was unaffected by RR. The magnitude of RR-induced inhibition of Ca2+ channel phosphorylation did not differ appreciably at saturating or subsaturating concentrations of Ca2+ or calmodulin, and in the absence or presence of protein phosphatase inhibitors. In contrast to the effects of RR, low concentrations of ryanodine (0.25-1 microM) caused significant stimulation (up to approximately 50%) of Ca2+ channel phosphorylation but had no effect on Ca(2+)-ATPase and phospholamban phosphorylation. These findings suggest that interaction of RR with the ryanodine receptor induces a "nonphosphorylatable state" of the Ca(2+)-release channel, likely through a conformational change involving occlusion of the CaM kinase phosphorylation site. On the other hand, ryanodine binding to the receptor may serve to maintain an open, "phosphorylatable state" of the channel.
在心肌中，已知与肌浆网（SR）相关的一种钙/钙调蛋白依赖性蛋白激酶（CaM激酶）可使膜蛋白受磷蛋白、钙ATP酶和钙释放通道（兰尼碱受体）磷酸化。受磷蛋白和钙ATP酶的磷酸化被认为可刺激肌浆网对钙的摄取，但钙通道磷酸化的功能后果尚未明确。在本研究中，我们研究了肌浆网钙释放抑制剂钌红（RR）和肌浆网钙释放激活剂兰尼碱（亚微摩尔浓度）对兔心肌肌浆网中CaM激酶介导的钙循环蛋白磷酸化的影响。在37℃下，将肌浆网与RR（5 - 30 microM）孵育3分钟，导致内源性膜相关CaM激酶对钙通道磷酸化的显著抑制（高达85%）（15±2 microM RR时抑制50%）。外源性添加的多功能αCaM激酶II对钙通道的磷酸化也同样受到RR的抑制。RR对内源性和外源性CaM激酶对钙ATP酶的磷酸化仅产生适度抑制（25 - 30%），而受磷蛋白的磷酸化不受RR影响。在钙或钙调蛋白饱和或不饱和浓度下，以及在存在或不存在蛋白磷酸酶抑制剂的情况下，RR诱导的钙通道磷酸化抑制程度没有明显差异。与RR的作用相反，低浓度的兰尼碱（0.25 - 1 microM）可显著刺激（高达约50%）钙通道磷酸化，但对钙ATP酶和受磷蛋白的磷酸化没有影响。这些发现表明，RR与兰尼碱受体的相互作用可能通过涉及CaM激酶磷酸化位点封闭的构象变化诱导钙释放通道的“不可磷酸化状态”。另一方面，兰尼碱与受体的结合可能有助于维持通道的开放“可磷酸化状态”。
