Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation

SLC6A14 (solute carrier family 6 member 14) is an amino acid transporter, driven by Na+ and Cl− co-transport, whose structure, function, and molecular and kinetic mechanism have not been well characterized. Its broad substrate selectivity, including neutral and cationic amino acids, differentiates it from other SLC6 family members, and its proposed involvement in nutrient transport in several cancers suggest that it could become an important drug target. In the present study, we investigated SLC6A14 function and its kinetic mechanism after expression in human embryonic kidney (HEK293) cells, including substrate specificity and voltage dependence under various ionic conditions. We applied rapid solution exchange, voltage jumps, and laser photolysis of caged alanine, allowing sub-millisecond temporal resolution, to study SLC6A14 steady state and pre-steady state kinetics. The results highlight the broad substrate specificity and suggest that extracellular chloride enhances substrate transport but is not required for transport. As in other SLC6 family members, Na+ binding to the substrate-free transporter (or conformational changes associated with it) is electrogenic and is likely rate limiting for transporter turnover. Transient current decaying with a time constant of <1ms is also observed after rapid amino acid application, both in forward transport and homoexchange modes, indicating a slightly electrogenic, but fast and not rate-limiting substrate translocation step. Our results, which are consistent with kinetic modeling, suggest rapid transporter turnover rate and substrate translocation with faster kinetics compared with other SLC6 family members. Together, these results provided novel information on the SLC6A14 transport cycle and mechanism, expanding our understanding of SLC6A14 function.

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure–Function Relationships in Mammalian SLC6 Transporters

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional–structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts.

A three-sodium-to-glycine stoichiometry shapes the structural relationships of ATB0,+ with GlyT2 and GlyT1 in the SLC6 family

GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na+:Cl-, respectively. However, ATB0,+ stoichiometry that specifies its driving force and electrogenicity remains unsettled. Using the reversal potential slope method, here we demonstrate that ATB0,+-mediated glycine transport is coupled to 3 Na+ and 1 Cl- and has a charge coupling of 2.1 e/glycine. ATB0,+ behaves as a unidirectional transporter with limited e and exchange capabilities. Analysis and computational modeling of the pre-steady-state charge movement reveal higher sodium affinity of the apo-ATB0,+, and a locking trap preventing Na+ loss at depolarized potentials. A 3 Na+/ 1 Cl- stoichiometry substantiates ATB0;+ concentrative-uptake and trophic role in cancers and rationalizes its structural proximity with GlyT2 despite their divergent substrate specificity. Analysis and computational modeling of the pre-steady-state charge movement reveal higher sodium affinity of the apo-ATB0,+, and a locking trap preventing Na+ loss at depolarized potentials. A 3 Na+/ 1 Cl- stoichiometry substantiates ATB0,+ concentrative-uptake and trophic role in cancers and rationalizes its structural proximity with GlyT2 despite their divergent substrate specificity.

Role of transmembrane helix 6 in substrate recognition of the amino acid transporter MhsT

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics

The treatment of chronic pain is poorly managed by current analgesics, and there is a need for new classes of drugs. We recently developed a series of bioactive lipids that inhibit the human glycine transporter GlyT2 (SLC6A5) and provide analgesia in animal models of pain. Here, we have used functional analysis of mutant transporters combined with molecular dynamics simulations of lipid-transporter interactions to understand how these bioactive lipids interact with GlyT2. This study identifies a novel extracellular allosteric modulator site formed by a crevice between transmembrane domains 5, 7, and 8, and extracellular loop 4 of GlyT2. Knowledge of this site could be exploited further in the development of drugs to treat pain, and to identify other allosteric modulators of the SLC6 family of transporters.

Serotonin transport in the 21st century

Serotonin (5-hydroxytryptamine [5-HT]) is accumulated within nerve endings by the serotonin transporter (SERT), which terminates its extracellular action and provides cytoplasmic 5-HT for refilling of synaptic vesicles. SERT is the target for many antidepressant medications as well as psychostimulants such as cocaine and ecstasy (3,4-methylenedioxymethamphetamine). SERT belongs to the SLC6 family of ion-coupled transporters and is structurally related to several other transporter families. SERT was studied in the 1970s and 1980s using membrane vesicles isolated from blood platelets. These studies led to a proposed stoichiometry of transport that has been challenged by high-resolution structures of SERT and its homologues and by studies of SERT electrophysiology. Here, we review the original evidence alongside more recent structural and electrophysiological evidence. A self-consistent picture emerges with surprising insights into the ion fluxes that accompany 5-HT transport.

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2

Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na+ and Cl−. Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4). Here, we posited that the transport cycle of individual SLC6 transporters reflects the physiological requirements they operate under. We tested this hypothesis by analyzing the transport cycle of glycine transporter 1 (GlyT1/SLC6A9) and glycine transporter 2 (GlyT2/SLC6A5). GlyT2 is the only SLC6 family member known to translocate glycine, Na+, and Cl− in a 1:3:1 stoichiometry. We analyzed partial reactions in real time by electrophysiological recordings. Contrary to monoamine transporters, both GlyTs were found to have a high transport capacity driven by rapid return of the empty transporter after release of Cl− on the intracellular side. Rapid cycling of both GlyTs was further supported by highly cooperative binding of cosubstrate ions and substrate such that their forward transport mode was maintained even under conditions of elevated intracellular Na+ or Cl−. The most important differences in the transport cycle of GlyT1 and GlyT2 arose from the kinetics of charge movement and the resulting voltage-dependent rate-limiting reactions: the kinetics of GlyT1 were governed by transition of the substrate-bound transporter from outward- to inward-facing conformations, whereas the kinetics of GlyT2 were governed by Na+ binding (or a related conformational change). Kinetic modeling showed that the kinetics of GlyT1 are ideally suited for supplying the extracellular glycine levels required for NMDA receptor activation.

How to rescue misfolded SERT, DAT and NET: targeting conformational intermediates with atypical inhibitors and partial releasers

Point mutations in the coding sequence for solute carrier 6 (SLC6) family members result in clinically relevant disorders, which are often accounted for by a loss-of-function phenotype. In many instances, the mutated transporter is not delivered to the cell surface because it is retained in the endoplasmic reticulum (ER). The underlying defect is improper folding of the transporter and is the case for many of the known dopamine transporter mutants. The monoamine transporters, i.e. the transporters for norepinephrine (NET/SLC6A2), dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4), have a rich pharmacology; hence, their folding-deficient mutants lend themselves to explore the concept of pharmacological chaperoning. Pharmacochaperones are small molecules, which bind to folding intermediates with exquisite specificity and scaffold them to a folded state, which is exported from the ER and delivered to the cell surface. Pharmacochaperoning of mutant monoamine transporters, however, is not straightforward: ionic conditions within the ER are not conducive to binding of most typical monoamine transporter ligands. A collection of compounds exists, which are classified as atypical ligands because they trap monoamine transporters in unique conformational states. The atypical binding mode of some DAT inhibitors has been linked to their anti-addictive action. Here, we propose that atypical ligands and also compounds recently classified as partial releasers can serve as pharmacochaperones.

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Neurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na+ binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na+ release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids.