Calcineurin-mediated protein dephosphorylation in brain nerve terminals regulates the release of glutamate.
In response to Ca2+ entry, several prominent brain nerve terminal phosphoproteins undergo dephosphorylation, but the relation between dephosphorylation and neurotransmitter release is unknown. Using the immunosuppressants cyclosporin A (CsA) and L-683,590 (FK-520) to inhibit specifically the Ca2+/calmodulin-dependent protein phosphatase calcineurin, we demonstrate here that Ca(2+)-dependent dephosphorylation in isolated rat brain nerve terminals (synaptosomes) is mediated by calcineurin. Pretreatment with micromolar CsA resulted in a 76-95% inhibition of stimulation-induced decreases in 32P-labeled dynamin (previously referred to as dephosphin), a phosphoprotein of M(r) = 145,000 (145-kDa protein), and a phosphoprotein of M(r) = 170,000 (170-kDa protein). Pretreatment with FK-520 also inhibited Ca(2+)-dependent dephosphorylation. Using hypotonic lysates of 32P-labeled synaptosomes, the addition of Ca2+ plus calmodulin, but not either agent alone, induced dynamin dephosphorylation. CsA and FK-520 had little to no effect on the release of glutamate induced by either K(+)-depolarization or the Ca2+ ionophore ionomycin. In contrast, calcineurin inhibition led to a substantial enhancement of glutamate release evoked by the K(+)-channel blocker 4-aminopyridine, an agent whose action most closely mimics physiological stimulation. Calcineurin inhibition had no effect on stimulation-induced changes in synaptosomal Ca2+ levels. Based on our findings, we hypothesize that Ca(2+)-dependent protein dephosphorylation resulting from calcineurin activation during physiological stimulation limits neurotransmitter release from brain nerve terminals, perhaps being dependent upon cyclic repolarization of the membrane during stimulation.
Nichols
R A
RA
Department of Pharmacology, Medical College of Pennsylvania, Philadelphia 19129.
Suplick
G R
GR
Brown
J M
JM
D015761:4-Aminopyridine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology*; D002474:Cell-Free System; D002540:Cerebral Cortex / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D034281:Dynamins; D004988:Ethers, Cyclic / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009411:Nerve Endings / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000378:metabolism / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects; D051381:Rats; D009435:Synaptic Transmission / Q000187:drug effects; D013574:Synaptosomes / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors
D002148:Calmodulin-Binding Proteins; D004988:Ethers, Cyclic; D005971:Glutamates; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D015221:Potassium Channels; D019319:Okadaic Acid; D016572:Cyclosporine; C058028:immunomycin; D015761:4-Aminopyridine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D020558:GTP Phosphohydrolases; D034281:Dynamins; D011188:Potassium; D002118:Calcium; D016559:Tacrolimus