Abstract
The vesicular monoamine transporters (VMATs) package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. One isoform occurs in the adrenal gland (VMAT1) and another in the brain (VMAT2). To assess their potential for regulation, we have investigated the phosphorylation of the VMATs. Using heterologous expression in Chinese hamster ovary, PC12, and COS cells, we find that rat VMAT2, but not VMAT1, is constitutively phosphorylated. Phosphoamino acid analysis indicates that this phosphorylation occurs on serine residues, and the analysis of VMAT1-VMAT2 chimeras and site-directed mutagenesis localize the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of VMAT2. Since these residues occur in an acidic region, we tested the ability of the acidotropic kinases casein kinase I (CKI) and casein kinase II (CKII) to phosphorylate bacterial fusion proteins containing the carboxyl terminus of VMAT2. Purified CKI and CKII phosphorylate the wild-type carboxyl terminus of VMAT2, but not a double mutant with both serines 512 and 514 replaced by alanine. The protein kinase inhibitor CKI-7 and unlabeled GTP both block in vitro phosphorylation by cell homogenates, indicating a role for CKII and possibly CKI in vivo. Both kinases phosphorylate the VMAT2 fusion protein to a much greater extent than a similar fusion protein containing the carboxyl terminus of VMAT1, consistent with differential phosphorylation of the two transporters observed in intact cells. These results provide the first demonstration of phosphorylation of a vesicular neurotransmitter transporter and a potential mechanism for differential regulation of the two VMATs.
Highlights
For classical neurotransmitters such as the monoamines, synaptic transmission involves two distinct transport activities
We have detected phosphorylation of the ϳ55-kDa form of VMAT2, but not VMAT1, in COS cells (Fig. 2B). These results indicate that VMAT2, but not VMAT1, undergoes constitutive phosphorylation in a variety of cell types
To assess the potential for regulation of vesicular monoamine transport, we have studied the phosphorylation state of the transport proteins and determined that the ϳ55-kDa form of the brain monoamine transporter VMAT2, but not the adrenal gland transporter VMAT1, undergoes constitutive phosphorylation in intact cells
Summary
For classical neurotransmitters such as the monoamines, synaptic transmission involves two distinct transport activities. Plasma membrane transport and vesicular neurotransmitter transport differ in their bioenergetic mechanism and sensitivity to drugs. Monoamine transport across the plasma membrane differs from vesicular transport in terms of pharmacology. Molecular cloning has recently identified several transport proteins responsible for packaging classical neurotransmitters including the monoamines into secretory vesicles [3, 4]. Transport of monoamines across the plasma membrane undergoes regulation by phosphorylation. Activation of Ca2ϩ- and phospholipid-dependent kinase by phorbol esters inhibits plasma membrane serotonin transport [20, 21]. The transport of neurotransmitter into synaptic vesicles may be regulated by phosphorylation. The mechanism of inhibition remains unclear, and the phosphorylation of a vesicular neurotransmitter transporter has not yet been demonstrated
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