Carboxyl Residues Required for Transport by a Vesicular Monoamine Transporter Homolog from Brevibacillus Brevis (BbMAT)

Abstract

The uptake and storage of neurotransmitters in synaptic vesicles is carried out by neurotransmitter transporters from, e.g., the vesicular monoamine transporter (VMAT) family, which allow regulated release of monoamines from the presynaptic neuron into the synaptic cleft. Phylogenetic analysis reveals that VMAT proteins are evolutionarily related to bacterial multidrug transporters involved in antibiotic resistance. In fact, VMATs are responsible for sequestering toxic substrates such as MPP+ into vesicles, away from their primary site of action. To understand the molecular mechanisms of transport by VMAT, we characterized a homologous transporter from Brevibacillus brevis (BbMAT) using molecular modeling and biochemical approaches. First, we built a homology model of BbMAT based on the crystal structure of YajR, a putative proton-driven MFS transporter from Escherichia coli. As expected, the model contains 12 transmembrane (TM) helices, arranged in two domains of 6 TMs each, which are related by 2-fold pseudo-symmetry around an axis perpendicular to the membrane between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the TM segments that line the central cavity. The activity of BbMAT coupled to a proton gradient was assessed biochemically, demonstrating that the transport process is electrogenic and therefore that at least two protons are exchanged for each substrate. The six residues exposed to the aqueous cavity according to the model were then mutated either alone or in combination, and these mutants were tested for their ability to confer resistance to ethidium and acriflavine. The results show that only two residues, D25 (TM1) and E229 (TM7), are essential for proton-coupled transport in BbMAT. We conclude that BbMAT is a paradigm for the study of multidrug and neurotransmitter transport by VMATs.