Jan-Philipp Machtens

Mechanisms of Anion Conduction by Coupled Glutamate Transporters

Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.

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.

Induced fit substrate binding to an archeal glutamate transporter homologue

Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. GltPh, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) GltPh in detergent micelles. At low millimolar [Na+], the addition of l-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na+], the kinetics were dominated by a fast component [kobs,fast; KD (Na+) = 22 ± 3 mM, nHill = 1.7 ± 0.3] with values of kobs,fast rising in a saturable manner to ≈500 s−1 (at 6 °C) with increasing [l-aspartate]. The binding kinetics of l-aspartate differed from the binding kinetics of two alternative substrates: l-cysteine sulfinic acid and d-aspartate. l-cysteine sulfinic acid bound with higher affinity than l-aspartate but involved lower saturating rates, whereas the saturating rates after d-aspartate binding were higher. Thus, after the association of two Na+ to the empty transporter, GltPh binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for GltPh than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

Background: GLT-1c and EAAT5 are two excitatory amino acid transporters co-expressed in retinal neurons. Results: GLT-1c and EAAT5 differ in glutamate and Na+ affinity, individual transport rates as well as in unitary anion current amplitudes. Conclusion: GLT-1c and EAAT5 are optimized to fulfill different physiological tasks. Significance: Identification of unitary channel properties underlying separate anion conductances associated with GLT-1c and EAAT5. In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as “excitatory amino acid transporters (EAATs).” Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na+ and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na+ and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.

Regulation of Glial Glutamate Transporters by C-terminal Domains

Excitatory amino acid transporter 2 (EAAT2) is a high affinity glutamate transporter predominantly expressed in astroglia. Human EAAT2 encompasses eight transmembrane domains and a 74-amino acid C-terminal domain that resides in the cytoplasm. We examined the role of this region by studying various C-terminal truncations and mutations using heterologous expression in mammalian cells, whole-cell patch clamp recording and confocal imaging. Removal of the complete C terminus (K498X EAAT2) results in loss of function because of intracellular retention of truncated proteins in the cytoplasm. However, a short stretch of amino acids (E500X EAAT2) within the C terminus results in correctly processed transporters. E500X reduced glutamate transport currents by 90%. Moreover, the voltage and substrate dependence of E500X EAAT2 anion currents was significantly altered. WT and mutant EAAT2 anion channels are modified by external Na+ in the presence as well as in the absence of l-glutamate. Whereas Na+ stimulates EAAT2 anion currents in the presence of l-glutamate, increased [Na+] reduces such currents without glutamate. In cells internally dialyzed with Na+, WT, and truncated EAAT2 display comparable Na+ dependence. With K+ as main internal cation, E500X drastically increased the apparent dissociation constant for external Na+. The effects of E500X can be represented by a kinetic model that allows translocation of the empty transporter from the outward- to the inward-facing conformation and stabilization of the inward-facing conformation by internal K+. Our results demonstrate that the C terminus modifies the glutamate uptake cycle, possibly affecting the movements of the translocation domain of EAAT2 glutamate transporter.

Molecular physiology of EAAT anion channels

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by a family of five glutamate transporters, the so-called excitatory amino acid transporters (EAAT1–5). EAATs are prototypic members of the growing number of dual-function transport proteins: they are not only glutamate transporters, but also anion channels. Whereas the mechanisms underlying secondary active glutamate transport are well understood at the functional and at the structural level, mechanisms and cellular roles of EAAT anion conduction have remained elusive for many years. Recently, molecular dynamics simulations combined with simulation-guided mutagenesis and experimental analysis identified a novel anion-conducting conformation, which accounts for all experimental data on EAAT anion currents reported so far. We here review recent findings on how EAATs accommodate a transporter and a channel in one single protein.

Substrate-dependent Gating of Anion Channels Associated with Excitatory Amino Acid Transporter 4

EAAT glutamate transporters do not only function as secondary-active glutamate transporters but also as anion channels. EAAT anion channel activity depends on transport substrates. For most isoforms, it is negligible without external Na+ and increased by external glutamate. We here investigated gating of EAAT4 anion channels with various cations and amino acid substrates using patch clamp experiments on a mammalian cell line. We demonstrate that Li+ can substitute for Na+ in supporting substrate-activated anion currents, albeit with changed voltage dependence. Anion currents were recorded in glutamate, aspartate, and cysteine, and distinct time and voltage dependences were observed. For each substrate, gating was different in external Na+ or Li+. All features of voltage-dependent and substrate-specific anion channel gating can be described by a simplified nine-state model of the transport cycle in which only amino acid substrate-bound states assume high anion channel open probabilities. The kinetic scheme suggests that the substrate dependence of channel gating is exclusively caused by differences in substrate association and translocation. Moreover, the voltage dependence of anion channel gating arises predominantly from electrogenic cation binding and membrane translocation of the transporter. We conclude that all voltage- and substrate-dependent conformational changes of the EAAT4 anion channel are linked to transitions within the transport cycle.

Neutralizing Aspartate 83 Modifies Substrate Translocation of Excitatory Amino Acid Transporter 3 (EAAT3) Glutamate Transporters

Background: Neutralizing a conserved aspartate between TM2 and TM3 affects gating of EAAT anion channels. Results: Voltage clamp fluorometry demonstrates that the analogous mutation alters EAAT3 substrate translocation. Conclusion: Altered substrate translocation is sufficient to explain anion channel gating in D83A EAAT3. Significance: Dissection of transport and anion channel gating defines an intimate relationship between transporter translocation and anion channel opening. 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.

Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations

Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92u2009±u20093% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function.

Noise analysis to study unitary properties of transporter-associated ion channels

Excitatory amino acid transporters (EAATs) do not only mediate secondary-active glutamate uptake but also function as anion channels. We recently used macroscopic current recordings and noise analysis to determine unitary current amplitudes of anion channels associated with a neuronal EAAT isoform, EAAT4. We found that, at symmetrical NO3-, EAAT4 anion channels exhibit a single channel conductance of ~1 pS in the absence as well as in the presence of glutamate. These results indicate that glutamate increases EAAT4 anion currents by modifying exclusively open probabilities, however, leaves unitary current amplitudes unaffected. Noise analysis has been developed for ion channels with a single conductance state and limitations might ensue when using this approach for transporter-associated ion channels. We here performed stochastic simulations of EAAT transporter-associated anion channels and noise analysis of simulated currents to assess the reliability and possible limitations of this technique in studying this special class of ion channels.