Neutralizing aspartate 83 modifies substrate translocation of excitatory amino acid transporter 3 (EAAT3) glutamate transporters

Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission by removing glutamate from the synaptic cleft into neuronal and glial cells. EAATs are not only secondary active glutamate transporters but also function as anion channels. Gating of EAAT anion channels is tightly coupled to transitions within the glutamate uptake cycle, resulting in Na(+)- and glutamate-dependent anion currents. A point mutation neutralizing a conserved aspartic acid within the intracellular loop close to the end of transmembrane domain 2 was recently shown to modify the substrate dependence of EAAT anion currents. To distinguish whether this mutation affects transitions within the uptake cycle or directly modifies the opening/closing of the anion channel, we used voltage clamp fluorometry. Using three different sites for fluorophore attachment, V120C, M205C, and A430C, we observed time-, voltage-, and substrate-dependent alterations of EAAT3 fluorescence intensities. The voltage and substrate dependence of fluorescence intensities can be described by a 15-state model of the transport cycle in which several states are connected to branching anion channel states. D83A-mediated changes of fluorescence intensities, anion currents, and secondary active transport can be explained by exclusive modifications of substrate translocation rates. In contrast, sole modification of anion channel opening and closing is insufficient to account for all experimental data. We conclude that D83A has direct effects on the glutamate transport cycle and that these effects result in changed anion channel function.     

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.     

Molecular Determinants for Functional Differences between Alanine-Serine-Cysteine Transporter 1 and Other Glutamate Transporter Family Members

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter Glt_Ph. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and Glt_Ph. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and Glt_Ph, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and Glt_Ph. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of Glt_Ph, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.     

Protein kinase C (PKC)-promoted endocytosis of glutamate transporter GLT-1 requires ubiquitin ligase Nedd4-2-dependent ubiquitination but not phosphorylation

Glutamate transporter-1 (GLT-1) is the main glutamate transporter in the central nervous system, and its concentration severely decreases in neurodegenerative diseases. The number of transporters in the plasma membrane reflects the balance between their insertion and removal, and it has been reported that the regulated endocytosis of GLT-1 depends on its ubiquitination triggered by protein kinase C (PKC) activation. Here, we identified serine 520 of GLT-1 as the primary target for PKC-dependent phosphorylation, although elimination of this serine did not impair either GLT-1 ubiquitination or endocytosis in response to phorbol esters. In fact, we present evidence indicating that the ubiquitin ligase Nedd4-2 mediates the PKC-dependent ubiquitination and down-regulation of GLT-1. Overexpression of Nedd4-2 increased the ubiquitination of the transporter and promoted its degradation. Moreover, phorbol myristate acetate enhanced Nedd4-2 phosphorylation and the formation of GLT-1·Nedd4-2 complexes, whereas siRNA knockdown of Nedd4-2 prevented ubiquitination, endocytosis, and the concomitant decrease in GLT-1 activity triggered by PKC activation. These results indicate that GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter.     

Proteome Analysis and Conditional Deletion of the EAAT2 Glutamate Transporter Provide Evidence against a Role of EAAT2 in Pancreatic Insulin Secretion in Mice

Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or β-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously.     

Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing

The dysregulation of the dopaminergic system is implicated in multiple neurological and neuropsychiatric disorders such as Parkinson disease and drug addiction. The primary target of psychostimulants such as amphetamine and methamphetamine is the dopamine transporter (DAT), the major regulator of extracellular dopamine levels in the brain. However, the behavioral and neurophysiological correlates of methamphetamine and amphetamine administration are unique from one another, thereby suggesting these two compounds impact dopaminergic neurotransmission differentially. We further examined the unique mechanisms by which amphetamine and methamphetamine regulate DAT function and dopamine neurotransmission; in the present study we examined the impact of extracellular and intracellular amphetamine and methamphetamine on the spontaneous firing of cultured midbrain dopaminergic neurons and isolated DAT-mediated current. In dopaminergic neurons the spontaneous firing rate was enhanced by extracellular application of amphetamine > dopamine > methamphetamine and was DAT-dependent. Amphetamine > methamphetamine similarly enhanced DAT-mediated inward current, which was sensitive to isosmotic substitution of Na⁺ or Cl⁻ ion. Although isosmotic substitution of extracellular Na⁺ ions blocked amphetamine and methamphetamine-induced DAT-mediated inward current similarly, the removal of extracellular Cl⁻ ions preferentially blocked amphetamine-induced inward current. The intracellular application of methamphetamine, but not amphetamine, prevented the dopamine-induced increase in the spontaneous firing of dopaminergic neurons and the corresponding DAT-mediated inward current. The results reveal a new mechanism for methamphetamine-induced dysregulation of dopaminergic neurons.     

Mutating a Conserved Proline Residue within the Trimerization Domain Modifies Na+ Binding to Excitatory Amino Acid Transporters and Associated Conformational Changes

Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3.     

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357–418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357–418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357–418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.     

Hetero-oligomerization of neuronal glutamate transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.     

Structure and Function of Sodium-coupled GABA and Glutamate Transporters

Neurotransmitter transporters are key elements in the termination of the synaptic actions of the neurotransmitters. They use the energy stored in the electrochemical ion gradients across the plasma membrane of neurons and glial cells for uphill transport of the transmitters into the cells surrounding the synapse. Therefore specific transporter inhibitors can potentially be used as novel drugs for neurological disease. Sodium-coupled neurotransmitter transporters belong to either of two distinct families. The glutamate transporters belong to the SLC1 family, whereas the transporters of the other neurotransmitters belong to the SLC6 family. An exciting and recent development is the emergence of the first high-resolution structures of archeal and bacterial members belonging to these two families. In this review the functional results on prototypes of the two families, the GABA transporter GAT-1 and the glutamate transporters GLT-1 and EAAC1, are described and discussed within the perspective provided by the novel structures.     

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp¹¹²) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.     

Role of Glutamate and GABA Transporters in Development of Pentylenetetrazol-Kindling

Kindling is a form of epileptogenesis that can be induced with pentylenetetrazol (PTZ). We undertook this study to evaluate the contribution of glutamate and GABA transporters to the process of PTZ kindling. Rats were injected i.p. three times per week with PTZ (40 mg/kg) until they were fully kindled. In rats who achieved full kindling, measurement of hippocampal glutamate and GABA transporters within 24 h by western blot showed that GLAST, GLT-1, and EAAC1 were elevated significantly. However, fully kindled rats at 30 days after their last seizure had no change in either glutamate or GABA transporters proteins. These sequential observations suggest that glutamate transporters may contribute to the occurrence of seizures, but were not associated with maintenance of epileptogenesis. During this experiment, we collected data from animals that had kindled easily and animals who were resistant to kindling. Easily-kindled rats reached full kindling with less than five injections of PTZ. Kindling resistant animals failed to achieve full kindling even after administration of 12 consecutive injections of PTZ. Levels of EAAC1 and GAT-1 in easily-kindled rats were decreased by 30% when compared to kindling resistant animals at 30 days after the last PTZ injection. Since decreased EAAC1 and GAT-1 would diminish GABA function, less quantity of these proteins would appear to be associated with the convulsive threshold at the beginning of kindling development. We wonder if glutamate and GABA transporters might be operant in a convulsion threshold set factor or as a pace factor for kindling.     

Single Expressed Glycine Receptor Domains Reconstitute Functional Ion Channels without Subunit-specific Desensitization Behavior

Cys loop receptors are pentameric arrangements of independent subunits that assemble into functional ion channels. Each subunit shows a domain architecture. Functional ion channels can be reconstituted even from independent, nonfunctional subunit domains, as shown previously for GlyRα1 receptors. Here, we demonstrate that this reconstitution is not restricted to α1 but can be transferred to other members of the Cys loop receptor family. A nonfunctional GlyR subunit, truncated at the intracellular TM3–4 loop by a premature stop codon, can be complemented by co-expression of the missing tail portion of the receptor. Compared with α1 subunits, rescue by domain complementation was less efficient when GlyRα3 or the GABA_A/C subunit ρ1 was used. If truncation disrupted an alternative splicing cassette within the intracellular TM3–4 loop of α3 subunits, which also regulates receptor desensitization, functional rescue was not possible. When α3 receptors were restored by complementation using domains with and without the spliced insert, no difference in desensitization was found. In contrast, desensitization properties could even be transferred between α1/α3 receptor chimeras harboring or lacking the α3 splice cassette proving that functional rescue depends on the integrity of the alternative splicing cassette in α3. Thus, an intact α3 splicing cassette in the TM3–4 loop environment is indispensable for functional rescue, and the quality of receptor restoration can be assessed from desensitization properties.     

Mechanism of Cation Binding to the Glutamate Transporter EAAC1 Probed with Mutation of the Conserved Amino Acid Residue Thr101

The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na⁺ ions, one H⁺ ion, and one glutamate molecule into the cell, in exchange for one K⁺ ion. Na⁺ binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na⁺] dependence of this conductance as a read-out of Na⁺ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr¹⁰¹, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na⁺ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1_WT, in the T101A mutant this [Na⁺] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na⁺ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na⁺ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr¹⁰¹ and Asp³⁶⁷. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.     

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.     

Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

Complex Regulation of Voltage-dependent Activation and Inactivation Properties of Retinal Voltage-gated Cav1.4 L-type Ca2+ Channels by Ca2+-binding Protein 4 (CaBP4)

Cav1.4 L-type Ca²⁺ channels are crucial for synaptic transmission in retinal photoreceptors and bipolar neurons. Recent studies suggest that the activity of this channel is regulated by the Ca²⁺-binding protein 4 (CaBP4). In the present study, we explored this issue by examining functional effects of CaBP4 on heterologously expressed Cav1.4. We show that CaBP4 dramatically increases Cav1.4 channel availability. This effect crucially depends on the presence of the C-terminal ICDI (inhibitor of Ca²⁺-dependent inactivation) domain of Cav1.4 and is absent in a Cav1.4 mutant lacking the ICDI. Using FRET experiments, we demonstrate that CaBP4 interacts with the IQ motif of Cav1.4 and that it interferes with the binding of the ICDI domain. Based on these findings, we suggest that CaBP4 increases Cav1.4 channel availability by relieving the inhibitory effects of the ICDI domain on voltage-dependent Cav1.4 channel gating. We also functionally characterized two CaBP4 mutants that are associated with a congenital variant of human night blindness and other closely related nonstationary retinal diseases. Although both mutants interact with Cav1.4 channels, the functional effects of CaBP4 mutants are only partially preserved, leading to a reduction of Cav1.4 channel availability and loss of function. In conclusion, our study sheds new light on the functional interaction between CaBP4 and Cav1.4. Moreover, it provides insights into the mechanism by which CaBP4 mutants lead to loss of Cav1.4 function and to retinal disease.     

Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer

The aspartate transporter from Pyrococcus horikoshii (Glt_Ph) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of Glt_Ph provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting Glt_Ph into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of Glt_Ph were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of Glt_Ph revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.     

Impact of Recovery from Desensitization on Acid-sensing Ion Channel-1a (ASIC1a) Current and Response to High Frequency Stimulation

ASIC1a is a neuronal sodium channel activated by external H⁺ ions. To date, all the characterization of ASIC1a has been conducted applying long H⁺ stimuli lasting several seconds. Such experimental protocols weaken and even silence ASIC1a currents to repetitive stimulation. In this work, we examined ASIC1a currents by methods that use rapid application and removal of H⁺. We found that brief H⁺ stimuli, <100 ms, even if applied at high frequency, prevent desensitization thereby generate full and steady peak currents of human ASIC1a. Kinetic analysis of recovery from desensitization of hASIC1a revealed two desensitized states: short- and long-lasting with time constants of τ_Ds ≤0.5 and τ_Dl = 229 s, while in chicken ASIC1a the two desensitized states have similar values τ_D 4.5 s. It is the large difference in stability of the two desensitized states that makes hASIC1a desensitization more pronounced and complex than in cASIC1a. Furthermore, recovery from desensitization was unrelated to cytosolic variations in pH, ATP, PIP₂, or redox state but was dependent on the hydrophobicity of key residues in the first transmembrane segment (TM1). In conclusion, brief H⁺-stimuli maintain steady the magnitude of peak currents thereby the ASIC1a channel is well poised to partake in high frequency signals in the brain.     

20-Hydroxyeicosatetraenoic acid (20-HETE) is a novel activator of transient receptor potential vanilloid 1 (TRPV1) channel

TRPV1 is a member of the transient receptor potential ion channel family and is gated by capsaicin, the pungent component of chili pepper. It is expressed predominantly in small diameter peripheral nerve fibers and is activated by noxious temperatures >42 °C. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A/4F-derived metabolite of the membrane phospholipid arachidonic acid. It is a powerful vasoconstrictor and has structural similarities with other TRPV1 agonists, e.g. the hydroperoxyeicosatetraenoic acid 12-HPETE, and we hypothesized that it may be an endogenous ligand for TRPV1 in sensory neurons innervating the vasculature. Here, we demonstrate that 20-HETE both activates and sensitizes mouse and human TRPV1, in a kinase-dependent manner, involving the residue Ser(502) in heterologously expressed hTRPV1, at physiologically relevant concentrations.