2.A.22 The Neurotransmitter:Sodium Symporter (NSS) Family

Members of the NSS family (SLC59) catalyze uptake of a variety of neurotransmitters, amino acids, osmolytes and related nitrogenous substances by a solute:Na⁺ symport mechanism (Rudnick et al. 2013). Sometimes Cl^- is cotransported, and some exhibit a K⁺ dependency. The human dopamine transporter probably co-transports the positively charged or zwitterionic dopamine species with 2Na⁺ and 1Cl^-. The human betaine/GABA transporter cotransports 3Na⁺ and 1 or 2Cl^- with one molecule of betaine or GABA. Two different glycine transporters, GlyT1 (TC #2.A.22.2.2) and GlyT2 (TC #2.A.22.2.6), cotransport glycine with 2Na⁺ and 3Na⁺, respectively as well as 1Cl^-. Most characterized members are from animals, but bacterial and archaeal homologues have been sequenced, and one bacterial homologue, TnaT of Symbiobacterium thermophilum, TC #2.A.22.5.2, has been shown to be a Na⁺-dependent tryptophan uptake permease with high affinity (145 nM) (Androutsellis-Theotokis et al., 2003) while a second is a tyrosine-specific Na⁺ symporter. Eukaryotic NSS proteins are generally of 600-800 amino acyl residues in length and possess 12 putative transmembrane helical spanners, but about 70% of prokaryotic homologues have 11 TMSs (Quick et al., 2006).  Several NSS family members have been characterized from marine invertebrates (Kinjo et al. 2013). Paudel et al. 2021 determined the effect of 24 different synthetic 4-benzylpiperidine carboxamides on the reuptake of serotonin, norepinephrine, and dopamine (DA). They identified (1) critical structural features contributing to the selectivity of a molecule for each of the monoamine transporters, (2) critical residues on the compounds that bound to the transporters, and (3) the functional role of a DA reuptake inhibitor in regulating D2R function. 1, 5-disubstituted tetrazoles are also monoamine neurotransmitter reuptake inhibitors (Paudel et al. 2021).

 Neurotransmitter: sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. In eukaryotic NSS, chloride is transported together with the neurotransmitter. However, transport by the bacterial homologues LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions. Zomot et al, (2007) found that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (γ-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux (but not of exchange) was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) similarly resulted in chloride-independent transport. The reciprocal mutations in LeuT and Tyt1 rendered substrate binding and/or uptake by these bacterial NSS chloride dependent. Their data indicated that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.

Evidence supports the conclusion that some members of the NSS family are dimers while others are monomers, and still others can be oligomeric depending on their localization. Thus, the glycine transporter is monomeric in the plasma membrane but oligomeric when intracellular. Both the serotonin and dopamine transporters may be dimeric. In the latter case, the extracellular end of TMS6 may be at a symmetrical dimer interface (Hastrup et al., 2001).  In mammals, several isoforms of these transporters (e.g., DAT and NET) can be generated by tissue-specific alternative splicing (Sogawa et al. 2010).  Shared dynamics of LeuT superfamily members and allosteric differentiation by structural irregularities and multimerization have been analyzed and reviewed (Ponzoni et al. 2018).

Tavoulari et al. (2011) described conversion of the Cl^- -independent prokaryotic tryptophan transporter TnaT (2.A.22.4.1) to a fully functional Cl^- -dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in a serotonin transporter (SERT; 2.A.22.1.1) provided direct evidence for the involvement of each of the proposed residues in Cl^- coordination. In both SERT and TnaT-D268S, Cl^- and Na⁺ mutually increase each other's potency, consistent with am electrostatic interaction through adjacent binding sites.

Several members of the NSS family have been shown to exhibit channel-like properties under certain experimental conditions. Thus, sizable unitary ionic currents have been reported for membrane patches containing either the γ-aminobutyrate, norepinephrine or serotonin transporter. In the presence of Zn²⁺ (10 μM), the dopamine transporter (DAT) catalyzes uncoupled Cl^- conductance (Meinild et al., 2004). Channel-like currents have also been measured for mammalian Na⁺/H⁺/K⁺-coupled glutamate transporters of the DAACS family (TC #2.A.23). Evidence shows that these channels can accommodate neurotransmitters as well as inorganic ions. One of these, CAATCH1 (TC #2.A.22.2.4) can function as an amino acid-gated cation (K⁺ and Na⁺) channel (Quick and Stevens, 2001). Different amino acids (pro, thr, met) differentially affect the state probability of the channel. These observations suggest that, as has been demonstrated for carriers of a few other families, neurotransmitter transporters can be induced to function as voltage-gated channels.

The GABA transporter, GAT-1 (TC #2.A.23.3.2), can catalyze channel-like fluxes of Li⁺ and K⁺. Mutations in TMS1 can lock the permease in the 'cation leak' mode (Kanner, 2003). The leak in the G63C (but not the G63S) mutant could be blocked by addition of membrane impermeable sulfhydryl reagents, suggesting that this position is accessible from the external aqueous medium. Thus, TMS1 contains determinants of both Na⁺-coupled GABA transport and the cation leak.

Cocaine and related drugs act by inhibiting clearance of released monoamine neurotransmitters from the synaptic cleft. Cocaine inhibits uptake of serotonin via SERT, dopamine via DAT, and norepinephrine via NET. Cocaine binds with high affinity to all three transporters, exhibiting competitive inhibition with the monoamine substrates, probably by binding to the active sites (Rasmussen et al., 2001). Dual inhibition of serotonin and norepinephrine transporters (hSERT and hNET) (serotonin-norepinephrine reuptake inhibitors, SNRIs) gives greatly improved efficacy and tolerability for treating major depressive disorder (MDD) compared with selective reuptake inhibitors (Xue et al. 2018).

The differential expression patterns and physiological roles of the glycine transporter subtypes have been exploited in the development of novel transport inhibitors to treat schizophrenia (GLYT1 inhibitors). GLYT1 transports glycine and also the N-methyl derivative of glycine, sarcosine, whereas GLYT2 only transports glycine. Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts as a coagonist with L-glutamate on the N-methyl-D-aspartate subtypes of glutamate receptors. Glycine transporters regulate glycine concentrations within both inhibitory and excitatory synapses. The GLYT1 subtypes of glycine transporters are expressed in glial cells surrounding both excitatory and inhibitory synapses, whereas the GLYT2 subtypes of glycine transporters are expressed in presynaptic inhibitory glycinergic neurons (Vandenberg et al. 2007).

There are two Na⁺/Cl^- -dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations, and these transporters show differences in substrate selectivity and blocker sensitivity. Differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2 (Vandenberg et al., 2007).

The crystal structure of a bacterial member of the NSS family has been determined complexed to leucine and 2 Na⁺ (Yamashita et al., 2005). The protein core consists of the first ten of the 12 TMSs with segments 1-5 and 6-10 exhibiting a pseudo-2-fold axis in the plane of the membrane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The binding pocket of LeuT contains two metal binding sites (Caplan et al., 2008). The first ion in site NA1 is directly coupled to the bound substrte (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Double ion occupancy of the binding pocket is required to ensure substrate coupling to Na⁺ (but not to Li⁺ or K⁺ cations). The presence of the ion in site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na⁺ in the case of double ion occupancy (Caplan et al., 2008).

Substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein (Zhao et al., 2011). In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine, alanine-induced dynamics are induced in the intracellular gate region of LeuT that directly correlate with transport efficiency. Thus, binding of a second substrate (S2) in the extracellular vestibule appears to act cooperatively with the primary substrate (S1) to control intracellular gating more than 30 Å away.

In the presence of Na⁺, the leucine-bound state of the invertebrate neutral amino acid transporter, KAAT1 of Manduca sexta (TC#2.A.22.2.5) is supposed to be relatively stable, while in the presence of K⁺, and at negative potentials, the progression of the leucine-bound form along the cycle is favoured. In this context, serine 308 appears to be important in allowing the change to the inward-facing conformation of the transporter following substrate binding, rather than in determining the binding specificity (Miszner et al., 2007). This lepidopteran neutral amino acid transporter has an unusual cation selectivity, being activated by K⁺ and Li⁺ in addition to Na⁺. Asp338 is essential for KAAT1 activation by K⁺ and for the coupling of amino acid transport to ion fluxes. Lys102 is likely to interact with Asp338 (Castagna et al., 2007). Asp338 corresponds to Asn286, a residue located in the Na⁺ binding site in the crystal structure of the NSS transporter LeuT. Lys102, interacting with Asp338, could contribute to the spatial organization of the KAAT1 cation binding site and the permeation pathway.

Transporters of the NSS family, or SLC6 family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits (Jayaraman et al. 2020). Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. It has been shown that oligomerization supports the activity of release-type psychostimulants. Single molecule microscopy revealed that the stoichiometry differs between individual members of the SLC6 family (Jayaraman et al. 2020). Moreover, handling of intracellular K⁺ determines the voltage dependence of plasmalemmal monoamine transporter function. Thus, DAT and NET differ from SERT in intracellular handling of K⁺. In DAT and NET, substrate uptake is voltage-dependent due to the transient nature of intracellular K⁺ binding, which precluded K⁺ antiport. SERT, however, antiports K⁺ and achieves voltage-independent transport (Bhat et al. 2021).

The generalized transport reaction for the members of this family is:

solute (out) + Na⁺ (out) → solute (in) + Na⁺ (in)