Research reportMurine vesicular monoamine transporter 2: molecular cloning and genomic structure
Introduction
Monoamine neurotransmitters including dopamine, norepinephrine, serotonin and histamine are accumulated into synaptic vesicles by the brain vesicular monoamine transporter (VMAT2) 13, 16, 17, 29, 33. VMAT2 pumps monoamines into vesicles using energy from proton co-transport down the pH gradient generated by the vesicular H+-ATPase 8, 13, 16, 17, 33. VMAT2 mRNA is localized to monoaminergic neurons and VMAT2 protein is largely localized to synaptic vesicular membranes 11, 25, 34, 40, 44. Indeed, VMAT2 is the principal currently identified gene whose expression is restricted to monoaminergic neurons.
VMAT2 function is important for proper packaging of monoamines for quantal, calcium-dependent vesicular release 13, 16, 17, 33. In addition, it may be important for sequestering toxins. The active metabolite, 1-methyl-4-phenyl-phenydium (MPP+), of the selective dopamine neuronal toxin N-methyl-1,2,3,6-tetrahydropyridine (MPTP) kills dopaminergic neurons or other cells that express the plasma membrane dopamine transporter (DAT) and causes a parkinsonian syndrome in vivo 1, 12, 18, 21, 35. Overexpression of VMAT, however, can suppress the toxicity of MPP+ by sequestering the toxin in vesicles away from presumed sites of cytoplasmic mitochondrial damage 24, 25, 26.
Amphetamines cause cytoplasmic release of monoamines from vesicular stores through disruption of the pH gradient across vesicular membranes 37, 39. The balance between this release and VMAT2-mediated vesicular re-uptake may be important for the non-quantal, calcium-independent extracellular dopamine release that modest amphetamine doses can yield, and for the dopaminergic toxicity that high-dose amphetamines can exert, perhaps through cytoplasmic oxidative stresses with dopamine oxidation and free radical production [38].
Recent molecular cloning studies have identified rat and human VMAT2 cDNAs, identifying this transporter as closely related to the VMAT1 cDNA clones expressed largely in adrenal chromaffin cells (VMAT1) and more distantly to the synaptic vesicular acetylcholine transporter 6, 7, 23, 25, 36, 40. However, no study to date has elucidated the structure and function of the VMAT2 gene. To seek insights into regulation of this protein so important for determining vesicular and cytoplasmic monoamine concentrations, to provide candidate sequences for cis acting elements necessary for monoaminergic-specific expression and to allow construction of transgenic mice in which altered expression of VMAT2 could allow better testing of postulated roles for VMAT2 in neurodegenerations and amphetamine actions, we now report elucidation of murine VMAT2 cDNAs and genomic sequences.
Section snippets
Identification of murine VMAT2 hybridization probe
Total RNA was isolated from murine midbrain tissue using RNAsolB™ (TEL-TEST). cDNA was prepared from 1 μg of this RNA using moloney murine leukemia virus (MMLV) reverse transcriptase and conditions suggested by the supplier (Clontech). cDNAs were amplified using the polymerase chain reaction (PCR) and two pairs of oligonucleotide primer sets: F1/R1 (5′-ATGGCCCTGAGCGATCTGGTGCTCTGCG-3′/5′-TGCTGGTAGCCTTGTGTGACTGCCCTCCTGG-3′), and F2/R2
cDNA cloning
Screening of 1×106 recombinant λ cDNA library plaques resulted in the isolation of two hybridizing clones. One of these clones, pCRIImVMAT2-cC contained a 4.2-kb insert. The nucleotide sequence of this clone revealed an open reading frame of 1551 bp, flanked by 35 bp of 5′ and 2.6 kb of 3′ untranslated region that included a polyadenylation signal (Fig. 1). The predicted 517 amino-acid protein showed 96, 92 and 60% amino-acid identify to rat and human VMAT2, and rat acetylcholine vesicular
Discussion
The present report documents sequences of the VMAT2 cDNA and gene of likely importance in identifying structural elements of the gene and its encoded protein, provides an assessment of the likelihood of presence of other closely related genomic sequences, identifies possible sequence elements that might contribute to monoamine-neuron-specific gene expression, and provides genomic elements useful for constructing transgenic mice with VMAT2 gene deletions and/or replacements.
The VMAT2 amino-acid
Acknowledgements
We thank Drs. David J. Vandenbergh and Christopher K. Surratt in our laboratory for helpful advice, Roxann Ingersoll in DNA Analysis Facility, Johns Hopkins University School of Medicine, for sequence analysis, Mary Jane Robinson and Angela Flood for help with the manuscript. We received support from the Intramural Research Program of the National Institute of Drug Abuse.
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