Genotype-phenotype correlation in CLCN4-related developmental and epileptic encephalopathy.

CLCN4-related disorder is a rare X-linked neurodevelopmental condition with a pathogenic mechanism yet to be elucidated. CLCN4 encodes the vesicular 2Cl-/H+ exchanger ClC-4, and CLCN4 pathogenic variants frequently result in altered ClC-4 transport activity. The precise cellular and molecular function of ClC-4 remains unknown; however, together with ClC-3, ClC-4 is thought to have a role in the ion homeostasis of endosomes and intracellular trafficking. We reviewed our research database for patients with CLCN4 variants and epilepsy, and performed thorough phenotyping. We examined the functional properties of the variants in mammalian cells using patch-clamp electrophysiology, protein biochemistry, and confocal fluorescence microscopy. Three male patients with developmental and epileptic encephalopathy were identified, with differing phenotypes. Patients #1 and #2 had normal growth parameters and normal-appearing brains on MRI, while patient #3 had microcephaly, microsomia, complete agenesis of the corpus callosum and cerebellar and brainstem hypoplasia. The p.(Gly342Arg) variant of patient #1 significantly impaired ClC-4's heterodimerization capability with ClC-3 and suppressed anion currents. The p.(Ile549Leu) variant of patient #2 and p.(Asp89Asn) variant of patient #3 both shift the voltage dependency of transport activation by 20 mV to more hyperpolarizing potentials, relative to the wild-type, with p.(Asp89Asn) favouring higher transport activity. We concluded that p.(Gly342Arg) carried by patient #1 and the p.(Ile549Leu) expressed by patient #2 impair ClC-4 transport function, while the p.(Asp89Asn) variant results in a gain-of-transport function; all three variants result in epilepsy and global developmental impairment, but with differences in epilepsy presentation, growth parameters, and presence or absence of brain malformations.

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.].

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.

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.

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.

Metabolic energy sensing by mammalian CLC anion/proton exchangers.

CLC anion/proton exchangers control the pH and [Cl- ] of the endolysosomal system that is essential for cellular nutrient uptake. Here, we use heterologous expression and whole-cell electrophysiology to investigate the regulation of the CLC isoforms ClC-3, ClC-4, and ClC-5 by the adenylic system components ATP, ADP, and AMP. Our results show that cytosolic ATP and ADP but not AMP and Mg2+ -free ADP enhance CLC ion transport. Biophysical analysis reveals that adenine nucleotides alter the ratio between CLC ion transport and CLC gating charge and shift the CLC voltage-dependent activation. The latter effect is suppressed by blocking the intracellular entrance of the proton transport pathway. We suggest, therefore, that adenine nucleotides regulate the internal proton delivery into the CLC transporter machinery and alter the probability of CLC transporters to undergo silent non-transporting cycles. Our findings suggest that the CBS domains in mammalian CLC transporters serve as energy sensors that regulate vesicular Cl- /H+ exchange by detecting changes in the cytosolic ATP/ADP/AMP equilibrium. Such sensing mechanism links the endolysosomal activity to the cellular metabolic state.

A Tripartite Interaction Among the Calcium Channel α1- and ß-Subunits and F-Actin Increases the Readily Releasable Pool of Vesicles and Its Recovery After Depletion.

Neurotransmitter release is initiated by the influx of Ca2+ via voltage-gated calcium channels. The accessory ß-subunit (CaVß) of these channels shapes synaptic transmission by associating with the pore-forming subunit (CaVα1) and up-regulating presynaptic calcium currents. Besides CaVα1, CaVß interacts with several partners including actin filaments (F-actin). These filaments are known to associate with synaptic vesicles (SVs) at the presynaptic terminals and support their translocation within different pools, but the role of CaVß/F-actin association on synaptic transmission has not yet been explored. We here study how CaVß4, the major calcium channel ß isoform in mamalian brain, modifies synaptic transmission in concert with F-actin in cultured hippocampal neurons. We analyzed the effect of exogenous CaVß4 before and after pharmacological disruption of the actin cytoskeleton and dissected calcium channel-dependent and -independent functions by comparing the effects of the wild-type subunit with the one bearing a double mutation that impairs binding to CaVα1. We found that exogenously expressed wild-type CaVß4 enhances spontaneous and depolarization-evoked excitatory postsynaptic currents (EPSCs) without altering synaptogenesis. CaVß4 increases the size of the readily releasable pool (RRP) of SVs at resting conditions and accelerates their recovery after depletion. The enhanced neurotransmitter release induced by CaVß4 is abolished upon disruption of the actin cytoskeleton. The CaVα1 association-deficient CaVß4 mutant associates with actin filaments, but neither alters postsynaptic responses nor the time course of the RRP recovery. Furthermore, this mutant protein preserves the ability to increase the RRP size. These results indicate that the interplay between CaVß4 and F-actin also support the recruitment of SVs to the RRP in a CaVα1-independent manner. Our studies show an emerging role of CaVß in determining SV maturation toward the priming state and its replenishment after release. We envision that this subunit plays a role in coupling exocytosis to endocytosis during the vesicle cycle.

VGLUT1 functions as a glutamate/proton exchanger with chloride channel activity in hippocampal glutamatergic synapses.

Glutamate is the major excitatory transmitter in the vertebrate nervous system. To maintain synaptic efficacy, recycling synaptic vesicles (SV) are refilled with glutamate by vesicular glutamate transporters (VGLUTs). The dynamics and mechanism of glutamate uptake in intact neurons are still largely unknown. Here, we show by live-cell imaging with pH- and chloride-sensitive fluorescent probes in cultured hippocampal neurons of wild-type and VGLUT1-deficient mice that in SVs VGLUT functions as a glutamate/proton exchanger associated with a channel-like chloride conductance. After endocytosis most internalized Cl- is substituted by glutamate in an electrically, and presumably osmotically, neutral manner, and this process is driven by both the Cl- gradient itself and the proton motive force provided by the vacuolar H+-ATPase. Our results shed light on the transport mechanism of VGLUT under physiological conditions and provide a framework for how modulation of glutamate transport via Cl- and pH can change synaptic strength.

Preferential association with ClC-3 permits sorting of ClC-4 into endosomal compartments.

ClC-4 is an intracellular Cl-/H+ exchanger that is highly expressed in the brain and whose dysfunction has been linked to intellectual disability and epilepsy. Here we studied the subcellular localization of human ClC-4 in heterologous expression systems. ClC-4 is retained in the endoplasmic reticulum (ER) upon overexpression in HEK293T cells. Co-expression with distinct ClC-3 splice variants targets ClC-4 to late endosome/lysosomes (ClC-3a and ClC-3b) or recycling endosome (ClC-3c). When expressed in cultured astrocytes, ClC-4 sorted to endocytic compartments in WT cells but was retained in the ER in Clcn3-/- cells. To understand the virtual absence of ER-localized ClC-4 in WT astrocytes, we performed association studies by high-resolution clear native gel electrophoresis. Although other CLC channels and transporters form stable dimers, ClC-4 was mostly observed as monomer, with ClC-3-ClC-4 heterodimers being more stable than ClC-4 homodimers. We conclude that unique oligomerization properties of ClC-4 permit regulated targeting of ClC-4 to various endosomal compartment systems via expression of different ClC-3 splice variants.

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions.

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.

Direct interaction of CaVß with actin up-regulates L-type calcium currents in HL-1 cardiomyocytes.

Expression of the ß-subunit (CaVß) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVß binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVß encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVß and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVß2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVß2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVß mediated L-type up-regulation, and CaVß-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVß that is directly involved in vesicular trafficking. We propose a model in which CaVß promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane.

Involvement of ClC-3 chloride/proton exchangers in controlling glutamatergic synaptic strength in cultured hippocampal neurons.

ClC-3 is a member of the CLC family of anion channels and transporters that localizes to early and late endosomes as well as to synaptic vesicles (SV). Its genetic disruption in mouse models results in pronounced hippocampal and retinal neurodegeneration, suggesting that ClC-3 might be important for normal excitatory and/or inhibitory neurotransmission in central neurons. To characterize the role of ClC-3 in glutamate accumulation in SV we compared glutamatergic synaptic transmission in cultured hippocampal neurons from WT and Clcn3-/- mice. In Clcn3-/- neurons the amplitude and frequency of miniature as well as the amplitudes of action-potential evoked EPSCs were significantly increased as compared to WT neurons. The low-affinity competitive AMPA receptor antagonist γ-DGG reduced the quantal size of synaptic events more effectively in WT than in Clcn3-/- neurons, whereas no difference was observed for the high-affinity competitive non-NMDA antagonist NBQX. Paired pulse ratios of evoked EPSCs were significantly reduced, whereas the size of the readily releasable pool was not affected by the genetic ablation of ClC-3. Electron microscopy revealed increased volumes of SV in hippocampi of Clcn3-/- mice. Our findings demonstrate that ClC-3 controls fast excitatory synaptic transmission by regulating the amount of neurotransmitter as well as the release probability of SV. These results provide novel insights into the role of ClC-3 in synaptic transmission and identify excessive glutamate release as a likely basis of neurodegeneration in Clcn3-/-.

The N-terminal domain tethers the voltage-gated calcium channel ß2e-subunit to the plasma membrane via electrostatic and hydrophobic interactions.

The ß-subunit associates with the α1 pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four ß-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, ß2a and ß2e, associate with the plasma membrane in the absence of the α1-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of ß2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which ß2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in ß2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic ß1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type ß2e, but not with a ß2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the ß2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the ß-subunit family that also sustains slowed inactivation.

ClC-3 is an intracellular chloride/proton exchanger with large voltage-dependent nonlinear capacitance.

The chloride/proton exchangers ClC-3, ClC-4 and ClC-5 are localized in distinct intracellular compartments and regulate their luminal acidity. We used electrophysiology combined with fluorescence pH measurements to compare the functions of these three transporters. Since the expression of WT ClC-3 in the surface membrane was negligible, we removed an N-terminal retention signal for standard electrophysiological characterization of this isoform. This construct (ClC-313-19A) mediated outwardly rectifying coupled Cl(-)/H(+) antiport resembling the properties of ClC-4 and ClC-5. In addition, ClC-3 exhibited large electric capacitance, exceeding the nonlinear capacitances of ClC-4 and ClC-5. Mutations of the proton glutamate, a conserved residue at the internal side of the protein, decreased ion transport but increased nonlinear capacitances in all three isoforms. This suggests that nonlinear capacitances in mammalian ClC transporters are regulated in a similar manner. However, the voltage dependence and the amplitudes of these capacitances differed strongly between the investigated isoforms. Our results indicate that ClC-3 is specialized in mainly performing incomplete capacitive nontransporting cycles, that ClC-4 is an effective coupled transporter, and that ClC-5 displays an intermediate phenotype. Mathematical modeling showed that such functional differences would allow differential regulation of luminal acidification and chloride concentration in intracellular compartments.

SNARE force synchronizes synaptic vesicle fusion and controls the kinetics of quantal synaptic transmission.

Neuronal communication relies on rapid and discrete intercellular signaling but neither the molecular mechanisms of the exocytotic machinery that define the timing of the action potential-evoked response nor those controlling the kinetics of transmitter release from single synaptic vesicles are known. Here, we investigate how interference with the putative force transduction between the complex-forming SNARE (soluble N-ethylamide-sensitive factor attachment protein receptor) domain and the transmembrane anchor of synaptobrevin II (SybII) affects action potential-evoked currents and spontaneous, quantal transmitter release at mouse hippocampal synapses. The results indicate that SybII-generated membrane stress effectively determines the kinetics of the action potential-evoked response and show that SNARE force modulates the concentration profile of cleft glutamate by controlling the rate of transmitter release from the single synaptic vesicle. Thus, multiple SybII actions determine the exquisite temporal regulation of neuronal signaling.