SLC18: Vesicular neurotransmitter transporters for monoamines and acetylcholine

Abstract

The exocytotic release of neurotransmitters requires active transport into synaptic vesicles and other types of secretory vesicles. Members of the SLC18 family perform this function for acetylcholine (SLC18A3, the vesicular acetylcholine transporter or VAChT) and monoamines such as dopamine and serotonin (SLC18A1 and 2, the vesicular monoamine transporters VMAT1 and 2, respectively). To date, no specific diseases have been attributed to a mutation in an SLC18 family member; however, polymorphisms in SLC18A1 and SLC18A2 may confer risk for some neuropsychiatric disorders. Additional members of this family include SLC18A4, expressed in insects, and SLC18B1, the function of which is not known. SLC18 is part of the Drug:H(+) Antiporter-1 Family (DHA1, TCID 2.A.1.2) within the Major Facilitator Superfamily (MFS, TCID 2.A.1).

Fig. 1

(A) Subcellular localization of SLC18A members. A canonical neuron showing a cell body and dendrites with an axonal process leading to the nerve terminal. Synaptic Vesicles (SVs) cluster at the nerve terminal and release neurotransmitter into the synapse when they fuse with the presynaptic plasma membrane. Large Dense Core Vesicles (LDCVs) localize more diffusely to sites throughout the cell body, dendrites and the nerve terminal and release classical as well as peptide neurotransmitters during exocytosis. SLC18A members can localize to one or both types of secretory vesicles. (B) Phylogenetic relationships. Phylogenetic tree with representative members of SLC18, including SLC18A1 (VMAT1), SLC18A2 (VMAT2), invertebrate VMAT, SLC18A3 (VAChT) and two newly defined subfamilies: SLC18A4, defined by the Drosophila gene portabella, and the novel subfamily SLC18B1. Note that portabella is present in additional insect species (not shown) but does not have an obvious vertebrate homolog. SLC18B1/C6orf192 homologs are present in multiple vertebrate and invertebrate species but not in Drosophila. Rat homologs of VMAT1, VMAT2 and VAChT overlap with Homo sapiens in this plot (not shown). Species represented in the diagram include Homo sapiens (Human), Gallus gallus (Chicken), Danio rerio (Zebrafish), Apis mellifera (Honeybee), Drosophila melanogaster (Drosophila) and Caenorhabditis elegans (C. elegans). Tree generated using the AllAll program of the Computational Biochemistry Research Group, Swiss Federal Institute of Technology, Zurich, Switzerland (http://www.cbrg.ethz.ch/services/AllAll) using protein sequences listed under the following accession numbers: NP_003046.2 (Human VAChT); NP_003045.2 (Human VMAT2); NP_001129163.1 (Human VMAT1); NP_439896.1 (Human C6orf192); NP_996865.1 (Chicken VAChT); XP_421782.2 (Chicken VMAT2); XP_428881.2 (Chicken VMAT1); XP_419737.3 (Chicken C6orf192); NP_001243154.1 (Zebrafish VMAT2); XP_002663262.2 (Zebrafish VMAT1), NP_001071018.1 (Zebrafish “probable vesicular acetylcholine transporter-A”); NP_001017841.1 (Zebrafish uncharacterized protein LOC550539 listed as C6orf192 in Jacobsson et al., 2010); AAF56164.2 (Drosophila CG10251/portabella); AAF55587.2 (Drosophila VAChT); AAX52708.1 (Drosophila VMAT, neural isoform DVMAT-A, see Greer et al., 2005), XP_001120960.2 (Honeybee VAChT); XP_392061.3 (Honey bee VMAT); XP_001122029.1, Honeybee portabella homolog annotated as “vesicular acetylcholine transporter unc-17-like”); XP_625161.2 (Honeybee C6orf192 homolog); CCA65520.1 (C. elegans VAChT, UNC-17 protein); NP_001024937.1 (C. elegans VMAT, CAT-1 protein); NP_741716.2 (hypothetical protein F55A4.8, listed as C. elegans C60orf192 in Jacobsson et al., 2010). Note that the sequence listed as “Drosophila C6orf192” in Jacobsson et al., 2010 is Drosophila VMAT (Greer et al., 2005) rather than a C6orf192 homolog. (C) Bioenergetics of SLC18A-dependent transport. Hydrolysis of ATP to ADP by a V-Type ATPase generates a proton gradient in the lumen of both SVs and LDCVs. The proton gradient (2 protons per cycle) drives transport of 1 molecule of positively charged neurotransmitter (NT) into the lumen of the vesicle via an antiport mechanism.

Fig. 2

Structure Function Studies of Transport and Trafficking. VMAT2 (A and C) and VAChT (B and D) contain 12 transmembrane domains. (A and B) Site directed mutagenesis has revealed the role of several residues for transport activity in VMAT2 (A) and VAChT (B). For VMAT2 these include aspartates (D) in the first and tenth membrane-bound domains required for transport activity, a charge-pair between residues in TM2 (K) and TM11 (D), and a proposed disulfide bond between cysteines (C) in two lumenal loops. Residues important for substrate affinity include a tyrosine (Y) in TM11 and an aspartate (D) in TM12. For VAChT, tryptophan (W) alanine (A) and phenyalanine (F) residues in TM8 and a cysteine (C) in TM10 are important for affinity to ACh. Conserved regions that contain glycine and/or proline (G/P) are required for transport activity and an aspartate (D) in TM10 is likely to be involved in proton exchange. Note that location of the residues in the cartoon is approximate and does not accurately reflect their precise position within each domain. (C and D) Membrane trafficking signals in VMAT2 (C) and VAChT (D). Glycosylation (“Y”) facilitates sorting of VMAT2 to Large Dense Core Vesicles (LVs). All other known sorting signals for vesicular transporters are encoded in their C-terminal, cytoplasmic domains. For VMAT2, a dileucine motif (IL) is required for endocytosis and sorting to Synaptic Like Microvesicles (SLMVs) in neuroendocrine cells. The VMAT2 endocytosis signal is part of a larger motif, which includes upstream glutamate residues (EE). The upstream glutamates are required for localizing VMAT2 to LVs. A phosphorylated “acidic cluster” at the extreme C-terminus of VMAT2 (DDEE[P]SE[P]SD, shown as [P]SD in panel (A)) also helps sort VMAT2 to LVs. (B) VAChT contains a dileucine motif (LL) required for endocytosis and sorting to SLMVs. However, a tyrosine-based motif (Y) may direct these trafficking events under some circumstances. A serine upstream of the dileucine motif in VAChT (SE) undergoes phosphorylation by PKC. Phosphorylation of the serine ([P]SE) may drive a portion of VAChT onto LVs.