Expanding the genetic and phenotypic relevance of CLCN4 variants in neurodevelopmental condition: 13 new patients.

OBJECTIVES: CLCN4 variations have recently been identified as a genetic cause of X-linked neurodevelopmental disorders. This study aims to broaden the phenotypic spectrum of CLCN4-related condition and correlate it with functional consequences of CLCN4 variants. METHODS: We described 13 individuals with CLCN4-related neurodevelopmental disorder. We analyzed the functional consequence of the unreported variants using heterologous expression, biochemistry, confocal fluorescent microscopy, patch-clamp electrophysiology, and minigene splicing assay. RESULTS: We identified five novel (p.R41W, p.L348V, p.G480R, p.R603W, c.1576 + 5G > A) and three known (p.T203I, p.V275M, p.A555V) pathogenic CLCN4 variants in 13 Chinese patients. The p.V275M variant is found at high frequency and seen in four unrelated individuals. All had global developmental delay (GDD)/intellectual disability (ID). Seizures were present in eight individuals, and 62.5% of them developed refractory epilepsy. Five individuals without seizures showed moderate to severe GDD/ID. Developmental delay precedes seizure onset in most patients. The variants p.R41W, p.L348V, and p.R603W compromise the anion/exchange function of ClC-4. p.R41W partially impairs ClC-3/ClC-4 association. p.G480R reduces ClC-4 expression levels and impairs the heterodimerization with ClC-3. The c.1576 + 5G > A variant causes 22 bp deletion of exon 10. CONCLUSIONS: We further define and broaden the clinical and mutational spectrum of CLCN4-related neurodevelopmental conditions. The p.V275M variant may be a potential hotspot CLCN4 variant in Chinese patients. The five novel variants cause loss of function of ClC-4. Transport dysfunction, protein instability, intracellular trafficking defect, or failure of ClC-4 to oligomerize may contribute to the pathophysiological events leading to CLCN4-related neurodevelopmental disorder.

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.

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.

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-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.

Zinc modulation of proton currents in a new voltage-gated proton channel suggests a mechanism of inhibition.

The HV 1 voltage-gated proton (HV 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of HV 1 is Zn2+ . Externally applied ZnCl2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn2+ and Cd2+ on HV 1 from Nicoletia phytophila, NpHV 1. We introduced mutations at potential Zn2+ coordination sites and measured Zn2+ inhibition in different extracellular pH, with Zn2+ concentrations up to 1000 µm. Zn2+ inhibition in NpHV 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn2+ in the S3-S4 linker. A simple Hodgkin Huxley model of NpHV 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpHV 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.

Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs.

Barttin is an accessory subunit of ClC-K chloride channels expressed in the kidney and the inner ear. Main functions of ClC-K/barttin channels are the generation of the cortico-medullary osmotic gradients in the kidney and the endocochlear potential in the inner ear. Mutations in the gene encoding barttin, BSND, result in impaired urinary concentration and sensory deafness. Barttin is predicted to be a two helical integral membrane protein that directly interacts with its ion channel in the membrane bilayer where it stabilizes the channel complex, promotes its incorporation into the surface membrane and leads to channel activation. It therefore is an attractive target to address fundamental questions of intermolecular communication within the membrane. However, so far inherent challenges in protein expression and stabilization prevented comprehensive in vitro studies and structural characterization. Here we demonstrate that cell-free expression enables production of sufficient quantities of an isotope-labeled barttin variant (I72X Barttin, capable to promote surface membrane insertion and channel activation) for NMR-based structural studies. Additionally, we established purification protocols as well as reconstitution strategies in detergent micelles and phospholipid bilayer nanodiscs. Stability, folding, and NMR data quality are reported as well as a suitable assignment strategy, paving the way to its structural characterization.

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.

Reduced Membrane Insertion of CLC-K by V33L Barttin Results in Loss of Hearing, but Leaves Kidney Function Intact.

In the mammalian ear, transduction of sound stimuli is initiated by K+ entry through mechano-sensitive channels into inner hair cells. K+ entry is driven by a positive endocochlear potential that is maintained by the marginal cell layer of the stria vascularis. This process requires basolateral K+ import by NKCC1 Na+-2Cl--K+ co-transporters as well as Cl- efflux through ClC-Ka/barttin or ClC-Kb/barttin channels. Multiple mutations in the gene encoding the obligatory CLC-K subunit barttin, BSND, have been identified in patients with Bartter syndrome type IV. These mutations reduce the endocochlear potential and cause deafness. As CLC-K/barttin channels are also expressed in the kidney, patients with Bartter syndrome IV typically also suffer from salt-wasting hyperuria and electrolyte imbalances. However, there was a single report on a BSND mutation that resulted only in deafness, but not kidney disease. We herein studied the functional consequences of another recently discovered BSND mutation that predicts exchange of valine at position 33 by leucine. We combined whole-cell patch clamp, confocal microscopy and protein biochemistry to analyze how V33L affects distinct functions of barttin. We found that V33L reduced membrane insertion of CLC-K/barttin complexes without altering unitary CLC-K channel function. Our findings support the hypothesis of a common pathophysiology for the selective loss of hearing due to an attenuation of the total chloride conductance in the stria vascularis while providing enough residual function to maintain normal kidney function.

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking.

ClC-K chloride channels are crucial for auditory transduction and urine concentration. Mutations in CLCNKB, the gene encoding the renal chloride channel hClC-Kb, cause Bartter syndrome type III, a human genetic condition characterized by polyuria, hypokalemia, and alkalosis. In recent years, several Bartter syndrome-associated mutations have been described that result in truncations of the intracellular carboxyl terminus of hClC-Kb. We here used a combination of whole-cell patch clamp, confocal imaging, co-immunoprecipitation, and surface biotinylation to study the functional consequences of a frequent CLCNKB mutation that creates a premature stop codon at Trp-610. We found that W610X leaves the association of hClC-Kb and the accessory subunit barttin unaffected, but impairs its regulation by barttin. W610X attenuates hClC-Kb surface membrane insertion. Moreover, W610X results in hClC-Kb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyl terminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltage-dependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyl terminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxyl-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyl terminus of hClC-Kb is not part of the binding site for barttin, but functionally modifies the interplay with barttin. The loss-of-activation of truncated hClC-Kb channels in heterologous expression systems fully explains the reduced basolateral chloride conductance in affected kidneys and the clinical symptoms of Bartter syndrome patients.

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor.

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3',5'-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 10(5) GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces "molecule noise." Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons.