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.

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability

Key points• ClC channels are double-barrelled channels with two ion conduction pathways per individual channel.• Substituting cysteine 277 by serine constitutively opens the common gate suggesting that this residue plays a major role in joint openings/closings of both protopores of ClC-1.• We studied here the functional consequences of two novel myotonia-associated mutations, C277R and C277Y, of human ClC-1 chloride channels.• C277Y not only modified the common gate, but also protopore gating.• C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates and thus decreases the absolute open probabilities of homodimeric hClC-1 channels to values below 3%.• C277Y reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence.• Our results explain the disease-causing effects and provide novel insights into the molecular processes underlying normal and pathologically altered function of muscle chloride channels.Abstract Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants' effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I − = NO 3 − > Br − > Cl − . Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects

ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1286-0) contains supplementary material, which is available to authorized users.

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

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 92 ± 3% 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.

ClC-K channels belong to the CLC family of chloride channels and chloride/proton antiporters. They contribute to sodium chloride reabsorption in Henle's loop of the kidney and to potassium secretion into the endolymph by the stria vascularis of the inner ear. Their accessory subunit barttin stabilizes the ClC-K/barttin complex, promotes its insertion into the surface membrane, and turns the pore-forming subunits into a conductive state. Barttin mutations cause Bartter syndrome type IV, a salt-wasting nephropathy with sensorineural deafness. Here, studying ClC-K/barttin channels heterologously expressed in MDCK-II and HEK293T cells with confocal imaging and patch-clamp recordings, we demonstrate that the eight-amino-acids-long barttin N terminus is required for channel trafficking and activation. Deletion of the complete N terminus (Δ2-8 barttin) retained barttin and human hClC-Ka channels in intracellular compartments. Partial N-terminal deletions did not compromise subcellular hClC-Ka trafficking but drastically reduced current amplitudes. Sequence deletions encompassing Thr-6, Phe-7, or Arg-8 in barttin completely failed to activate hClC-Ka. Analyses of protein expression and whole-cell current noise revealed that inactive channels reside in the plasma membrane. Substituting the deleted N terminus with a polyalanine sequence was insufficient for recovering chloride currents, and single amino acid substitutions highlighted that the correct sequence is required for proper function. Fast and slow gate activation curves obtained from rat V166E rClC-K1/barttin channels indicated that mutant barttin fails to constitutively open the slow gate. Increasing expression of barttin over that of ClC-K partially recovered this insufficiency, indicating that N-terminal modifications of barttin alter both binding affinities and gating properties.

Advances in structure-function analyses and computational biology have enabled a deeper understanding of how excitatory amino acid transporters (EAATs) mediate chloride permeation and substrate transport. However, the mechanism of structural coupling between these functions remains to be established. Using a combination of molecular modeling, substituted cysteine accessibility, electrophysiology and glutamate uptake assays, we identified a chloride-channeling conformer, iChS, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this iChS is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path (global); and repacking of a cluster of hydrophobic residues near the extracellular vestibule (local). Moreover, our results demonstrate that stabilization of iChS by chemical modifications favors anion channeling at the expense of substrate transport, suggesting a mutually exclusive regulation mediated by the movement of the flexible wall lining the two regions.