Abstract
Catechol-O-methyl transferase (COMT) is involved in the inactivation of dopamine in brain regions in which the dopamine transporter (DAT1) is sparsely expressed. The membrane-bound isoform of COMT (MB-COMT) is the predominantly expressed form in the mammalian central nervous system (CNS). It has been a matter of debate whether in neural cells of the CNS the enzymatic domain of MB-COMT is oriented toward the cytoplasmic or the extracellular compartment. Here we used live immunocytochemistry on cultured neocortical neurons and glial cells to investigate the expression and membrane orientation of native COMT and of transfected MB-COMT fused to green fluorescent protein (GFP). After live staining, COMT immunoreactivity was reliably detected in both neurons and glial cells after permeabilization, but not on unpermeabilized cells. Similarly, autofluorescence of COMT-GFP fusion protein and antibody fluorescence showed overlap only in permeabilized neurons. Our data provide converging evidence for an intracellular membrane orientation of MB-COMT in neurons and glial cells, suggesting the presence of a DAT1-independent postsynaptic uptake mechanism for dopamine, prior to its degradation via COMT.
Highlights
Catechol-O-methyl transferase (COMT, EC_number 2.1.1.6) is involved in the degradation of dopamine and other catecholamines by transferring a methyl group donated by S-adenosyl-methionine onto a phenolic hydroxyl group
Expression of native COMT in neurons and glial cells Co-staining with antibodies against COMT and microtubule-associated protein 2 (MAP2) or glial fibrillary acidic protein (GFAP) showed that COMT immunoreactivity was reliably detectable in both, cultured cortical neurons (Figures 1C,D,G,H) and glial cells (Figures 1K,L,O,P)
The intracellular localization and orientation of membrane-bound isoform of COMT (MB-COMT) in neurons and glial cells is critical for the understanding of the precise mechanism by which COMT influences dopamine availability in brain structures with low dopamine transporter activity
Summary
Catechol-O-methyl transferase (COMT, EC_number 2.1.1.6) is involved in the degradation of dopamine and other catecholamines by transferring a methyl group donated by S-adenosyl-methionine onto a phenolic hydroxyl group. Converging evidence from animal studies and human post mortem investigations raised the hypothesis that COMT is important for dopamine inactivation when dopamine uptake by the presynaptic dopamine transporter (DAT1) is inhibited or in brain regions in which DAT1 is sparsely expressed, such as the prefrontal cortex (PFC; Karoum et al, 1994; Sesack et al, 1998; Matsumoto et al, 2003). Previous studies have demonstrated that COMT is highly expressed in glial cells and perisynaptically in neurons (Rivett et al, 1983b; Karhunen et al, 1995a; Matsumoto et al, 2003). These studies do not provide information on the membrane orientation of the enzyme.
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