In monoaminergic cells, the neurotransmitter is accumulated into secretory or synaptic vesicles by a tetrabenazine-and reserpine-sensitive transporter, catalyzing an H+/monoamine antiport. The major vesicular monoamine transporter from bovine chromaffin cells was cloned, using sequences common to adrenal medulla and brain rat vesicular monoamine transporters. Its identity was confirmed by peptide sequences, determined from the purified protein. Surprisingly, the bovine adrenal medulla sequence, bVMAT,, is more related to the transporter from human and rat brain than to that from rat adrenal medulla. PCR amplifi~tion showed that bVMAT, is expressed in both adrenal medulla and brain, in contrast with the situation reported in rats, where distinct genes appear to be expressed in brain (SVAT or MAT, now renamed rVMAT& and in the adrenal medulla (CGAT, now renamed rVMA'I',). In bovine ~hromaffin cells, long-term depolarization by KCI resulted in an increase in the level of bVMAT, mRNA, in agreement with the previously observed increase in the transporter binding sites, suggesting that a coupiing between stimulation, secretion and synthesis changes the composition of the secretory granule membrane.
The monoamine transporter of the chromaffin granule membranes can be specifically labeled by the photoaffinity reagent 7-azido-8-[125I]iodoketanserin. The characteristics of the labeled protein have been investigated. Two-dimensional gel electrophoresis of the labeled membranes indicated a MW of about 70,000 and an isoelectric point ranging from 3.8 to 4.6. No clear protein spot was associated with the radioactive material, which migrated between glycoproteins GPII and GPIV. The diffuse aspect of the radioactive material indicated a heterogeneity, which was not modified after a second electrophoresis. This heterogeneity was, at least partially, due to glycosylation of the transporter; neuraminidase treatment increased the protein pI up to 6.3, whereas digestion with N-glycopeptidase markedly decreased the apparent MW, from 70,000 to 50,000. SDS-polyacrylamide gel electrophoresis showed that, at low acrylamide concentrations, the labeled material migrated more rapidly than predicted from the mobility of the markers of molecular weight, a behavior which indicated a marked hydrophobicity of the transporter. The labeled protein was purified to homogeneity by a combination of chromatography on DEAE-cellulose at pH 4.5, on immobilized wheat germ agglutinin, and on hydroxylapatite in the presence of SDS. During this purification, the specific radioactivity was increased by a factor of 300-500, with a yield of 10-20%.
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