Acetylcholine Receptor Stimulation Mediates Trafficking of the Dopamine and Glutamate Transporters through Activation of Protein Kinase C

Abstract

The dopamine transporter (DAT) clears neurotransmitter from the extracellular space and serves as an important regulator of signal amplitude and duration at sites of dopamine release. Similarly, the neuronal excitatory amino acid transporter 3 (EAAT3) plays an important role in the control of glutamatergic tone. Several different intracellular signaling pathways have been observed to modulate activity of these neurotransmitter transporters through regulation of the trafficking of the carriers to and from the cell surface. In this study, we investigated how the activation of protein kinase C (PKC) facilitates internalization of DAT and insertion of EAAT3 by addressing the endogenous cell-surface receptors that activate PKC and regulate the DAT and EAAT3 in vivo. We report here that stimulation of M1/M5 muscarinic receptors in midbrain cultures decreases the ability of dopamine neurons to transport dopamine through DAT. The cholinergic agonist, carbachol decreases DAT activity in primary cultures while the M1/M5-specific antagonist, pirenzepine, blocks these effects. The M3 antagonist, DAU 5884, has no effect, but a positive modulator of M5, VU 0238429, enhances the loss of DAT function in response to carbachol and acetylcholine. These data implicate M1/M5 receptors on dopamine neurons in the modulation of DAT function. Bisindolylmaleimide, a PKC inhibitor, blocks the effects of carbachol stimulation on dopamine uptake, indicating a role for PKC in muscarinic receptor-mediated DAT internalization. We designed a specific Gq inhibitor peptide that also blocks the effects of carbachol on DAT in primary cultures, confirming Gq as the G-protein that couples M1/M5 receptors to PKC activation in these cells. It has been established that the activity of the neuronal glutamate transporter EAAT3 increases in response to PMA treatment in the C6 glioma cell line. By total internal reflection fluorescence (TIRF) microscopy of a GFP-tagged EAAT3 we were able to confirm that this was due to an increase in EAAT3 present at the cell surface following PMA treatment. Carbachol activation of endogenous muscarinic receptors in the C6 glioma line also resulted in increased cell-surface expression of EAAT3. This increase was inhibited by co-application of the Gq inhibitor peptide indicating that the increase in EAAT3 activity depends upon Gq-coupled PKC signaling through M1/M5 receptors. In acute midbrain slices, biotinylation of cell-surface proteins showed that the decreases in dopamine transport mediated by muscarinic receptor stimulation the result of decreases in DAT carriers a cell surface. In addition, increases in surface expression of EAAT3 in the same experiment also appear to be modulated by M1/M5 receptors. These data indicate that stimulation of cholinergic receptors can lead to downregulation and upregulation of dopamine and glutamate transporters, respectively through alterations in their membrane trafficking.