Miles K, Audigier S S, Greengard P, Huganir R L
Department of Anatomy and Cell Biology, State University of New York Health Science Center at Brooklyn 11203.
J Neurosci. 1994 May;14(5 Pt 2):3271-9. doi: 10.1523/JNEUROSCI.14-05-03271.1994.
We have investigated the regulation of phosphorylation of the nicotinic ACh receptor (nAChR) in rat myotubes by the agonist carbamylcholine. Treatment of primary rat myotube cultures with carbamylcholine resulted in a 100% increase in phosphorylation of the nAChR gamma- (52 kDa) subunit and a 30% increase in phosphorylation of the nAChR delta- (62 kDa) and delta'- (66 kDa) subunits. These responses to carbamylcholine were dose dependent, with a half-maximal response occurring at 10 microM and a maximum response achieved within 2 min. Pretreatment of myotubes with d-tubocurare, but not with atropine, inhibited carbamylcholine-stimulated phosphorylation of the nAChR. Preincubation with open-channel blockers of the nAChR also inhibited phosphorylation of the nAChR induced by carbamylcholine. Depletion of extracellular calcium from myotube cultures prevented carbamylcholine-stimulated increases in nAChR phosphorylation whereas application of a calcium ionophore mimicked the effect of carbamylcholine on nAChR phosphorylation. Pretreatment of myotubes with TTX did not inhibit carbamylcholine-stimulated nAChR phosphorylation and potassium depolarization of myotubes had no effect on nAChR phosphorylation. Carbamylcholine increased nAChR phosphorylation to the same extent and with the same time course and subunit specificity as that induced by phorbol esters. However, chronic treatment of myotubes with phorbol esters that eliminated any subsequent phorbol ester-stimulated nAChR phosphorylation did not diminish the increase in nAChR phosphorylation induced by carbamylcholine. The calmodulin antagonist W7 was similarly unable to inhibit carbamylcholine-stimulated nAChR phosphorylation. These results suggest that the nAChR is a substrate for an uncharacterized protein kinase in situ, and that activity of this protein kinase is stimulated by calcium ions that permeate through the activated nAChR ion channel.(ABSTRACT TRUNCATED AT 250 WORDS)
我们研究了激动剂氨甲酰胆碱对大鼠肌管中烟碱型乙酰胆碱受体(nAChR)磷酸化的调节作用。用氨甲酰胆碱处理原代大鼠肌管培养物,导致nAChRγ-(52 kDa)亚基的磷酸化增加100%,nAChRδ-(62 kDa)和δ'-(66 kDa)亚基的磷酸化增加30%。这些对氨甲酰胆碱的反应呈剂量依赖性,在10 microM时出现半数最大反应,2分钟内达到最大反应。用d-筒箭毒碱预处理肌管,但不用阿托品预处理,可抑制氨甲酰胆碱刺激的nAChR磷酸化。用nAChR的开放通道阻滞剂预孵育也可抑制氨甲酰胆碱诱导的nAChR磷酸化。从肌管培养物中耗尽细胞外钙可阻止氨甲酰胆碱刺激的nAChR磷酸化增加,而应用钙离子载体可模拟氨甲酰胆碱对nAChR磷酸化的作用。用TTX预处理肌管不抑制氨甲酰胆碱刺激的nAChR磷酸化,肌管的钾离子去极化对nAChR磷酸化无影响。氨甲酰胆碱使nAChR磷酸化增加的程度、时间进程和亚基特异性与佛波酯诱导的相同。然而,用佛波酯长期处理肌管消除了随后任何佛波酯刺激的nAChR磷酸化,但并未减少氨甲酰胆碱诱导 的nAChR磷酸化增加。钙调蛋白拮抗剂W7同样无法抑制氨甲酰胆碱刺激的nAChR磷酸化。这些结果表明,nAChR是一种原位未鉴定的蛋白激酶的底物,并且这种蛋白激酶的活性受到通过活化的nAChR离子通道渗透的钙离子的刺激。(摘要截短于250字)