Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6.

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.

Dual-color Colocalization in Single-molecule Localization Microscopy to Determine the Oligomeric State of Proteins in the Plasma Membrane.

Determining the oligomeric state of membrane proteins is critical for understanding their function. However, traditional ex situ methods like clear native gel electrophoresis can disrupt protein subunit interactions during sample preparation. In situ methods such as stepwise photobleaching have limitations due to high expression levels and limitations of optical resolution in microscopy. Super-resolution microscopy techniques such as single-molecule localization microscopy (SMLM) have the potential to overcome these limitations, but the stochastic nature of signals can lead to miscounting due to over-expression, background noise, and temporal separation of signals. Additionally, this technique has limited application due to the limited selection of fluorescent labels and the demanding control of laser power. To address these issues, we developed a dual color colocalization (DCC) strategy that offers higher tolerance to background noise and simplifies data acquisition and processing for high-throughput and reliable counting. The DCC strategy was used to determine the oligomeric states of membrane proteins of the SLC17 and SLC26 family with SMLM, providing a robust and efficient method for studying protein interactions.

Vesicular glutamate transporters are H+-anion exchangers that operate at variable stoichiometry.

Vesicular glutamate transporters accumulate glutamate in synaptic vesicles, where they also function as a major Cl- efflux pathway. Here we combine heterologous expression and cellular electrophysiology with mathematical modeling to understand the mechanisms underlying this dual function of rat VGLUT1. When glutamate is the main cytoplasmic anion, VGLUT1 functions as H+-glutamate exchanger, with a transport rate of around 600 s-1 at -160 mV. Transport of other large anions, including aspartate, is not stoichiometrically coupled to H+ transport, and Cl- permeates VGLUT1 through an aqueous anion channel with unitary transport rates of 1.5 × 105 s-1 at -160 mV. Mathematical modeling reveals that H+ coupling is sufficient for selective glutamate accumulation in model vesicles and that VGLUT Cl- channel function increases the transport efficiency by accelerating glutamate accumulation and reducing ATP-driven H+ transport. In summary, we provide evidence that VGLUT1 functions as H+-glutamate exchanger that is partially or fully uncoupled by other anions.

Corrigendum: ClC-3 regulates the excitability of nociceptive neurons and is involved in inflammatory processes within the spinal sensory pathway.

[This corrects the article DOI: 10.3389/fncel.2022.920075.].

Determination of oligomeric states of proteins via dual-color colocalization with single molecule localization microscopy.

The oligomeric state of plasma membrane proteins is the result of the interactions between individual subunits and an important determinant of their function. Most approaches used to address this question rely on extracting these complexes from their native environment, which may disrupt weaker interactions. Therefore, microscopy techniques have been increasingly used in recent years to determine oligomeric states in situ. Classical light microscopy suffers from insufficient resolution, but super-resolution methods such as single molecule localization microscopy (SMLM) can circumvent this problem. When using SMLM to determine oligomeric states of proteins, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual molecules are counted based on a binomial distribution analysis of emission events detected within the same diffraction-limited volume. This strategy requires low background noise, a high recall rate for the fluorescent tag and intensive post-imaging data processing. To overcome these limitations, we developed a new method based on SMLM to determine the oligomeric state of plasma membrane proteins. Our dual-color colocalization (DCC) approach allows for accurate in situ counting even with low efficiencies of fluorescent protein detection. In addition, it is robust in the presence of background signals and does not require temporal clustering of localizations from individual proteins within the same diffraction-limited volume, which greatly simplifies data acquisition and processing. We used DCC-SMLM to resolve the controversy surrounding the oligomeric state of two SLC26 multifunctional anion exchangers and to determine the oligomeric state of four members of the SLC17 family of organic anion transporters.

Functional Characterization of CLCN4 Variants Associated With X-Linked Intellectual Disability and Epilepsy.

Early/late endosomes, recycling endosomes, and lysosomes together form the endo-lysosomal recycling pathway. This system plays a crucial role in cell differentiation and survival, and dysregulation of the endo-lysosomal system appears to be important in the pathogenesis of neurodevelopmental and neurodegenerative diseases. Each endo-lysosomal compartment fulfils a specific function, which is supported by ion transporters and channels that modify ion concentrations and electrical gradients across endo-lysosomal membranes. CLC-type Cl-/H+ exchangers are a group of endo-lysosomal transporters that are assumed to regulate luminal acidification and chloride concentration in multiple endosomal compartments. Heterodimers of ClC-3 and ClC-4 localize to various internal membranes, from the endoplasmic reticulum and Golgi to recycling endosomes and late endosomes/lysosomes. The importance of ClC-4-mediated ion transport is illustrated by the association of naturally occurring CLCN4 mutations with epileptic encephalopathy, intellectual disability, and behavioral disorders in human patients. However, how these mutations affect the expression, subcellular localization, and function of ClC-4 is insufficiently understood. We here studied 12 CLCN4 variants that were identified in patients with X-linked intellectual disability and epilepsy and were already characterized to some extent in earlier work. We analyzed the consequences of these mutations on ClC-4 ion transport, subcellular trafficking, and heterodimerization with ClC-3 using heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings. The mutations led to a variety of changes in ClC-4 function, ranging from gain/loss of function and impaired heterodimerization with ClC-3 to subtle impairments in transport functions. Our results suggest that even slight functional changes to the endosomal Cl-/H+ exchangers can cause serious neurological symptoms.

CLC Anion/Proton Exchangers Regulate Secretory Vesicle Filling and Granule Exocytosis in Chromaffin Cells.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Molecular Basis of Coupled Transport and Anion Conduction in Excitatory Amino Acid Transporters.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by excitatory amino acid transporters (EAATs) 1-5, a subfamily of glutamate transporters. The five proteins utilize a complex transport stoichiometry that couples glutamate transport to the symport of three Na+ ions and one H+ in exchange with one K+ to accumulate glutamate against up to 106-fold concentration gradients. They are also anion-selective channels that open and close during transitions along the glutamate transport cycle. EAATs belong to a larger family of secondary-active transporters, the SLC1 family, which also includes purely Na+- or H+-coupled prokaryotic transporters and Na+-dependent neutral amino acid exchangers. In recent years, molecular cloning, heterologous expression, cellular electrophysiology, fluorescence spectroscopy, structural approaches, and molecular simulations have uncovered the molecular mechanisms of coupled transport, substrate selectivity, and anion conduction in EAAT glutamate transporters. Here we review recent findings on EAAT transport mechanisms, with special emphasis on the highly conserved hairpin 2 gate, which has emerged as the central processing unit in many of these functions.

Mutations associated with epileptic encephalopathy modify EAAT2 anion channel function.

OBJECTIVE: Mutations in the gene solute carrier family member 1A2 (SLC1A2) encoding the excitatory amino acid transporter 2 (EAAT2) are associated with severe forms of epileptic encephalopathy. EAAT2 is expressed in glial cells and presynaptic nerve terminals and represents the main l-glutamate uptake carrier in the mammalian brain. It does not only function as a secondary active glutamate transporter, but also as an anion channel. How naturally occurring mutations affect these two transport functions of EAAT2 and how such alterations cause epilepsy is insufficiently understood. METHODS: Here we studied the functional consequences of three disease-associated mutations, which predict amino acid exchanges p.Gly82Arg (G82R), p.Leu85Pro (L85P), and p.Pro289Arg (P289R), by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings of EAAT2 l-glutamate transport and anion current. RESULTS: G82R and L85P exchange amino acid residues that contribute to the formation of the EAAT anion pore. They enlarge the pore diameter sufficiently to permit the passage of l-glutamate and thus function as l-glutamate efflux pathways. The mutation P289R decreases l-glutamate uptake, but increases anion currents despite a lower membrane expression. SIGNIFICANCE: l-glutamate permeability of the EAAT anion pore is an unexpected functional consequence of naturally occurring single amino acid substitutions. l-glutamate efflux through mutant EAAT2 anion channels will cause glutamate excitotoxicity and neuronal hyperexcitability in affected patients. Antagonists that selectively suppress the EAAT anion channel function could serve as therapeutic agents in the future.

ClC-3 regulates the excitability of nociceptive neurons and is involved in inflammatory processes within the spinal sensory pathway.

ClC-3 Cl-/H+ exchangers are expressed in multiple endosomal compartments and likely modify intra-endosomal pH and [Cl-] via the stoichiometrically coupled exchange of two Cl- ions and one H+. We studied pain perception in Clcn3-/- mice and found that ClC-3 not only modifies the electrical activity of peripheral nociceptors but is also involved in inflammatory processes in the spinal cord. We demonstrate that ClC-3 regulates the number of Na v and K v ion channels in the plasma membrane of dorsal root ganglion (DRG) neurons and that these changes impair the age-dependent decline in excitability of sensory neurons. To distinguish the role of ClC-3 in Cl-/H+ exchange from its other functions in pain perception, we used mice homozygous for the E281Q ClC-3 point mutation (Clcn3E281Q/E281Q ), which completely eliminates transport activity. Since ClC-3 forms heterodimers with ClC-4, we crossed these animals with Clcn4 -/- to obtain mice completely lacking in ClC-3-associated endosomal chloride-proton transport. The electrical properties of Clcn3 E281Q/E281Q /Clcn4-/- DRG neurons were similar to those of wild-type cells, indicating that the age-dependent adjustment of neuronal excitability is independent of ClC-3 transport activity. Both Clcn3-/- and Clcn3E281Q/E281Q /Clcn4 -/- animals exhibited microglial activation in the spinal cord, demonstrating that competent ClC-3 transport is needed to maintain glial cell homeostasis. Our findings illustrate how reduced Cl-/H+ exchange contributes to inflammatory responses and demonstrate a role for ClC-3 in the homeostatic regulation of neuronal excitability beyond its function in endosomal ion balance.

Excitatory Amino Acid Transporter EAAT5 Improves Temporal Resolution in the Retina.

Excitatory amino acid transporters (EAATs) remove glutamate from the synaptic cleft. In the retina, EAAT1 and EAAT2 are considered the major glutamate transporters. However, it has not yet been possible to determine how EAAT5 shapes the retinal light responses because of the lack of a selective EAAT5 blocker or EAAT5 knock-out (KO) animal model. In this study, EAAT5 was found to be expressed in a punctate manner close to release sites of glutamatergic synapses in the mouse retina. Light responses from retinae of wild-type (WT) and of a newly generated model with a targeted deletion of EAAT5 (EAAT5-/-) were recorded in vitro using multielectrode arrays (MEAs). Flicker resolution was considerably lower in EAAT5-/- retinae than in WT retinae. The close proximity to the glutamate release site makes EAAT5 an ideal tool to improve temporal information processing in the retina by controlling information transfer at glutamatergic synapses.

Glial Chloride Homeostasis Under Transient Ischemic Stress.

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl-]int) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl-]int are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl-]int in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl-]int, ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na+-K+-2Cl- cotransporter (NKCC1) and chloride outward transport (efflux) through K+-Cl- cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl-]int to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl-]int, whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl-]int was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl-]int in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl-]int in any of the tested glial cell types. However, [Cl-]int changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.

Dysregulation of Astrocyte Ion Homeostasis and Its Relevance for Stroke-Induced Brain Damage.

Ischemic stroke is a leading cause of mortality and chronic disability. Either recovery or progression towards irreversible failure of neurons and astrocytes occurs within minutes to days, depending on remaining perfusion levels. Initial damage arises from energy depletion resulting in a failure to maintain homeostasis and ion gradients between extra- and intracellular spaces. Astrocytes play a key role in these processes and are thus central players in the dynamics towards recovery or progression of stroke-induced brain damage. Here, we present a synopsis of the pivotal functions of astrocytes at the tripartite synapse, which form the basis of physiological brain functioning. We summarize the evidence of astrocytic failure and its consequences under ischemic conditions. Special emphasis is put on the homeostasis and stroke-induced dysregulation of the major monovalent ions, namely Na+, K+, H+, and Cl-, and their involvement in maintenance of cellular volume and generation of cerebral edema.

The molecular and phenotypic spectrum of CLCN4-related epilepsy.

OBJECTIVE: This study was undertaken to expand the phenotypic and genetic spectrum of CLCN4-related epilepsy and to investigate genotype-phenotype correlations. METHODS: We systematically reviewed the phenotypic and genetic spectrum of newly diagnosed and previously reported patients with CLCN4-related epilepsy. Three novel variants identified in four patients reported in this study were evaluated through in silico prediction and functional analysis by Western blot, immunofluorescence, and electrophysiological measurements. RESULTS: Epilepsy was diagnosed in 54.55% (24/44) of individuals with CLCN4-related disorders and was drug-resistant in most cases. Of 24 patients, 15 had epileptic encephalopathy and four died at an early age; 69.57% of patients had seizure onset within the first year of life. Myoclonic seizures are the most common seizure type, and 56.25% of patients presented multiple seizure types. Notably, seizure outcome was favorable in individuals with only one seizure type. All patients showed intellectual disability, which was severe in 65.22% of patients. Additional common features included language delay, behavioral disorders, and dysmorphic features. Five patients benefitted from treatment with lamotrigine. Most variants, which were mainly missense (79.17%), were inherited (70.83%). Whereas frameshift, intragenic deletion, or inherited variants were associated with milder phenotypes, missense or de novo variants led to more severe phenotypes. All evaluated CLCN4 variants resulted in loss of function with reduced ClC-4 currents. Nonetheless, genotype-phenotype relationships for CLCN4-related epilepsy are not straightforward, as phenotypic variability was observed in recurrent variants and within single families. SIGNIFICANCE: Pathogenic CLCN4 variants contribute significantly to the genetic etiology of epilepsy. The phenotypic spectrum of CLCN4-related epilepsy includes drug-resistant seizures, cognitive and language impairment, behavioral disorders, and congenital anomalies. Notably, the mutation type and the number of seizure types correlate with the severity of the phenotype, suggesting its use for clinical prognosis. Lamotrigine can be considered a therapeutic option.

Mechanisms Underlying Proton Release in CLC-type F-/H+ Antiporters.

The CLC family of anion channels and transporters includes Cl-/H+ exchangers (blocked by F-) and F-/H+ exchangers (or CLCFs). CLCFs contain a glutamate (E318) in the central anion-binding site that is absent in CLC Cl-/H+ exchangers. The X-ray structure of the protein from Enterococcus casseliflavus (CLCF-eca) shows that E318 tightly binds to F- when the gating glutamate (E118; highly conserved in the CLC family) faces the extracellular medium. Here, we use classical and DFT-based QM/MM metadynamics simulations to investigate proton transfer and release by CLCF-eca. After up to down movement of protonated E118, both glutamates combine with F- to form a triad, from which protons and F- anions are released as HF. Our results illustrate how glutamate insertion into the central anion-binding site of CLCF-eca permits the release of H+ to the cytosol as HF, thus enabling a net 1:1 F-/H+ stoichiometry.

Cellular Physiology and Pathophysiology of EAAT Anion Channels.

Excitatory amino acid transporters (EAATs) optimize the temporal resolution and energy demand of mammalian excitatory synapses by quickly removing glutamate from the synaptic cleft into surrounding neuronal and glial cells and ensuring low resting glutamate concentrations. In addition to secondary active glutamate transport, EAATs also function as anion channels. The channel function of these transporters is conserved in all homologs ranging from archaebacteria to mammals; however, its physiological roles are insufficiently understood. There are five human EAATs, which differ in their glutamate transport rates. Until recently the high-capacity transporters EAAT1, EAAT2, and EAAT3 were believed to conduct only negligible anion currents, with no obvious function in cell physiology. In contrast, the low-capacity glutamate transporters EAAT4 and EAAT5 are thought to regulate neuronal signaling as glutamate-gated channels. In recent years, new experimental approaches and novel animal models, together with the discovery of a human genetic disease caused by gain-of-function mutations in EAAT anion channels have enabled identification of the first physiological and pathophysiological roles of EAAT anion channels.