Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters.
Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.
Schonn
Jean-Sébastien
JS
CNRS UPR 1929, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France.
Desnos
Claire
C
Henry
Jean-Pierre
JP
Darchen
François
F
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D004298:Dopamine / Q000493:pharmacokinetics; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents / Q000378:metabolism* / Q000493:pharmacokinetics; D050484:Norepinephrine Plasma Membrane Transport Proteins; D016716:PC12 Cells / Q000187:drug effects; D010673:Pheochromocytoma / Q000378:metabolism*; D051381:Rats; D012110:Reserpine / Q000494:pharmacology; D022142:Secretory Vesicles / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism / Q000493:pharmacokinetics; D050486:Serotonin Plasma Membrane Transport Proteins; D027981:Symporters / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D018759:Adrenergic Uptake Inhibitors; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493226:Slc6a2 protein, rat; C493232:Slc6a4 protein, rat; D027981:Symporters; D012701:Serotonin; D012110:Reserpine; D004298:Dopamine