Renae M. Ryan

Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter

Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of GltPh, a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-β-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound α-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.

Mechanisms of Glutamate Transport

L-Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and plays important roles in a wide variety of brain functions, but it is also a key player in the pathogenesis of many neurological disorders. The control of glutamate concentrations is critical to the normal functioning of the central nervous system, and in this review we discuss how glutamate transporters regulate glutamate concentrations to maintain dynamic signaling mechanisms between neurons. In 2004, the crystal structure of a prokaryotic homolog of the mammalian glutamate transporter family of proteins was crystallized and its structure determined. This has paved the way for a better understanding of the structural basis for glutamate transporter function. In this review we provide a broad perspective of this field of research, but focus primarily on the more recent studies with a particular emphasis on how our understanding of the structure of glutamate transporters has generated new insights.

The uncoupled chloride conductance of a bacterial glutamate transporter homolog

Glutamate transporters (EAATs) are pivotal in mammalian synaptic transmission, tightly regulating synaptic levels of this excitatory neurotransmitter. In addition to coupled glutamate transport, the EAATs also show an uncoupled Cl− conductance, whose physiological importance has recently been demonstrated. Little is yet known about the molecular mechanism of chloride permeation. Here we show that GltPh, a bacterial EAAT homolog whose structure has been determined, displays an uncoupled Cl− conductance that can determine the rate of substrate uptake. A mutation analogous to one known to specifically affect Cl− movement in EAAT1 has similar effects on GltPh, suggesting that this protein is an excellent structural model for understanding Cl− permeation through the EAATs. We also observed an uncoupled Cl− conductance in another bacterial EAAT homolog but not in a homolog of the Na+/Cl−-coupled neurotransmitter transporters.

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAFWT (C8161 and WM852) and BRAFV600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAFWT and BRAFV600E melanoma.

Functional characterization of a Na+-dependent aspartate transporter from Pyrococcus horikoshii.

Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of GltPh, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described GltPh as an aspartate transporter driven by Na+ or a glutamate transporter driven by either Na+ or H+. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the GltPh transport. We confirm that GltPh is a Na+-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na+ ions. In contrast to the EAATs, transport via GltPh is independent of H+ and K+. We propose a kinetic model of transport in which at least two Na+ ions are coupled to the cotransport of each aspartate molecule by GltPh, and where an ion- and substrate-free transporter reorients to complete the transport cycle.

Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria

Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 and EAAC1) is the major epithelial transporter of glutamate and aspartate in the kidneys and intestines of rodents. Within the brain, SLC1A1 serves as the predominant neuronal glutamate transporter and buffers the synaptic release of the excitatory neurotransmitter glutamate within the interneuronal synaptic cleft. Recent studies have also revealed that polymorphisms in SLC1A1 are associated with obsessive-compulsive disorder (OCD) in early-onset patient cohorts. Here we report that SLC1A1 mutations leading to substitution of arginine to tryptophan at position 445 (R445W) and deletion of isoleucine at position 395 (I395del) cause human dicarboxylic aminoaciduria, an autosomal recessive disorder of urinary glutamate and aspartate transport that can be associated with mental retardation. These mutations of conserved residues impeded or abrogated glutamate and cysteine transport by SLC1A1 and led to near-absent surface expression in a canine kidney cell line. These findings provide evidence that SLC1A1 is the major renal transporter of glutamate and aspartate in humans and implicate SLC1A1 in the pathogenesis of some neurological disorders.

Position of the Third Na+ Site in the Aspartate Transporter GltPh and the Human Glutamate Transporter, EAAT1

Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na+ ions, one H+ and the counter-transport of one K+ ion. Transport by an archaeal homologue of the human glutamate transporters, GltPh, whose three dimensional structure is known is also coupled to three Na+ ions but only two Na+ ion binding sites have been observed in the crystal structure of GltPh. In order to fully utilize the GltPh structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of GltPh and accurately determine the number and location of Na+ ions coupled to transport. Several sites have been proposed for the binding of a third Na+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for GltPh and reveal a new site for the third Na+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in GltPh, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na+ ion in GltPh and EAAT1.

Effects of sumatriptan on rat medullary dorsal horn neurons

&NA; This study examined the cellular actions of the anti‐migraine drug sumatriptan, on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis. Sumatriptan inhibited the miniature EPSC (mEPSC) rate in a dose dependent fashion, with an EC50 of 250 nM. Sumatriptan (3 &mgr;M) inhibited the mEPSC rate by 36%, without altering the mEPSC amplitude. This effect was partially reversed by the 5HT1D specific antagonist BRL15572 (10 &mgr;M). In contrast, the 5HT1B agonist CP93129 (10 &mgr;m) did not alter the mEPSC rate. Furthermore, sumatriptan (3 &mgr;M) decreased the amplitude of electrically evoked EPSCs (eEPSC) by 40%. After incubating the slices in ketanserin (an antagonist which shows selectivity for 5HT1D over 5HT1B receptors) sumatriptan had little effect on eEPSC amplitude. In control conditions paired stimuli resulted in paired pulse depression (PPD; the ratio eEPSC2/eEPSC1=0.7±0.01), whilst in the presence of sumatriptan the PPD was blocked (ratio eEPSC2/eEPSC1=0.9±0.1). Sumatriptan produced no post‐synaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (−60 to −130 mV). RT‐PCR experiments revealed the presence of mRNA for both 5HT1D and 5HT1B receptor subtypes in the trigeminal ganglia and subnucleus caudalis. These data suggest that sumatriptan acts pre‐synaptically on trigeminal primary afferent central terminals to reduce the probability of release of glutamate, and that this action is mediated through 5HT1D receptors.

Prostaglandin E2 inhibits calcium current in two sub‐populations of acutely isolated mouse trigeminal sensory neurons

Prostaglandins are important mediators of pain and inflammation. We have examined the effects of prostanoids on voltage‐activated calcium currents (ICa) in acutely isolated mouse trigeminal sensory neurons, using standard whole cell voltage clamp techniques. Trigeminal neurons were divided into two populations based on the presence (Type 2) or absence (Type 1) of low voltage‐activated T‐type ICa. The absence of T‐type ICa is highly correlated with sensitivity to μ‐opioid agonists and the VR1 agonist capsaicin. In both populations of cells, high voltage‐activated ICa was inhibited by PGE2 with an EC50 of about 35 nm, to a maximum of 30 %. T‐type ICa was not inhibited by PGE2. Pertussis toxin pre‐treatment abolished the effects of PGE2 in Type 2 cells, but not in Type 1 cells, whereas treatment with cholera toxin prevented the effects of PGE2 in Type 1 cells, but not in Type 2 cells. Inhibition of ICa by PGE2 was associated with slowing of current activation and could be relieved with a large positive pre‐pulse, consistent with inhibition of ICa by G protein βγ subunits. Reverse transcription‐polymerase chain reaction of mRNA from trigeminal ganglia indicated that all four EP prostanoid receptors were present. However, in both Type 1 and Type 2 cells the effects of PGE2 were only mimicked by the selective EP3 receptor agonist ONO‐AE‐248, and not by selective agonists for EP1 (ONO‐DI‐004), EP2 (ONO‐AE1–259) and EP4 (ONO‐AE1–329) receptors. These data indicate that two populations of neurons in trigeminal ganglia differing in their calcium channel expression, sensitivity to μ‐opioids and capsaicin also have divergent mechanisms of PGE2‐mediated inhibition of calcium channels, with Gi/Go type G proteins involved in one population, and Gs type G proteins in the other.

Glycine transport inhibitors for the treatment of pain.

Opioids, local anesthetics, anticonvulsant drugs, antidepressants, and non-steroidal anti-inflammatory drugs (NSAIDs) are used to provide pain relief but they do not provide adequate pain relief in a large proportion of chronic pain patients and are often associated with unacceptable side effects. Inhibitory glycinergic neurotransmission is impaired in chronic pain states, and this provides a novel target for drug development. Inhibitors of the glycine transporter 2 (GlyT2) enhance inhibitory neurotransmission and show particular promise for the treatment of neuropathic pain. N-arachidonyl-glycine (NAGly) is an endogenous lipid that inhibits glycine transport by GlyT2 and also shows potential as an analgesic, which may be further exploited in drug development. In this review we discuss the role of glycine neurotransmission in chronic pain and future prospects for the use of glycine transport inhibitors in the treatment of pain.