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The nine-member CLC gene family of Cl− chloride-transporting membrane proteins is divided into plasma membrane-localized Cl− channels and endo-/lysosomal Cl−/H+ antiporters. Accessory proteins have been identified for ClC-K and ClC-2 channels and for the lysosomal ClC-7, but not the other CLCs. Here, we identified TMEM9 Domain Family Member B (TMEM9B), a single-span type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Co-expression of TMEM9B with ClC-3 or ClC-4 dramatically reduced transporter activity in Xenopus oocytes and transfected HEK cells. For ClC-3, TMEM9B also induced a slow component in the kinetics of the activation time course, suggesting direct interaction. Currents mediated by ClC-7 were hardly affected by TMEM9B, and ClC-1 currents were only slightly reduced, demonstrating specific interaction with ClC-3 and ClC-4. We obtained strong evidence for direct interaction by detecting significant Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with ClC-1 or ClC-7. The discovery of TMEM9B as a novel interaction partner of ClC-3 and ClC-4 might have important implications for the physiological role of these transporters in neuronal endosomal homeostasis and for a better understanding of the pathological mechanisms in CLCN3- and CLCN4-related pathological conditions. [ABSTRACT FROM AUTHOR]

Loss-, gain-of-function and mixed variants in SCN1A (Nav1.1 voltage-gated sodium channel) have been associated with a spectrum of neurologic disorders with different severity and drug-responsiveness. Most SCN1A variants are heterozygous changes occurring de novo or dominantly inherited; recessive inheritance has been reported in a few cases. Here, we report a family in which the biallelic inheritance of two novel SCN1A variants, N935Y and H1393Q, occurs in two siblings presenting with drug-responsive developmental and epileptic encephalopathy and born to heterozygous asymptomatic parents. To assess the genotype–phenotype correlation and support the treatment choice, HEK 293 cells were transfected with different combinations of the SCN1A WT and mutant cDNAs, and the resulting sodium currents were recorded through whole-cell patch-clamp. Functional studies showed that the N935Y and H1393Q channels and their combinations with the WT (WT + N935Y and WT + H1393Q) had current densities and biophysical properties comparable with those of their respective control conditions. This explains the asymptomatic condition of the probands' parents. The co-expression of the N935Y + H1393Q channels, mimicking the recessive inheritance of the two variants in siblings, showed ~20% reduced current amplitude compared with WT and with parental channels. This mild loss of Nav1.1 function may contribute in part to the disease pathogenesis, although other mechanisms may be involved. [ABSTRACT FROM AUTHOR]

ClC‐6 and ClC‐7 are closely related, intracellular Cl−/H+ antiporters belonging to the CLC family of channels and transporters. They localize to acidic late endosomes and lysosomes and probably function in ionic homeostasis of these contiguous compartments. ClC‐7 transport function requires association with the accessory protein Ostm1, whereas ClC‐6 transport does not. To elucidate their roles in endo‐lysosomes, we measured Cl−‐ and pH‐dependences of over‐expressed wild‐type ClC‐6 and ClC‐7, as well as disease‐associated mutants, using high‐resolution recording protocols. Lowering extracellular Cl− (corresponding to luminal Cl− in endo‐lysosomes) reduced ClC‐6 currents, whereas it increased transport activity of ClC‐7/Ostm1. Low extracellular Cl− activated ClC‐7/Ostm 1 under acidic extracellular conditions, as well as under conditions of low intracellular chloride. Activation is conserved in ClC‐7Y713C, a variant displaying disrupted PI(3,5)P2 inhibition. Detailed biophysical analysis of disease‐associated ClC‐6 and ClC‐7 gain‐of‐function (GoF) variants, ClC‐6Y553C and ClC‐7Y713C, and the ClC‐7Y577C and ClC‐6Y781C correlates, identified additional functional nuances distinguishing ClC‐6 and ClC‐7. ClC‐7Y577C recapitulated GoF produced by ClC‐6Y553C. ClC‐6Y781C displayed transport activation qualitatively similar to ClC‐7Y713C, although current density did not differ from that of wild‐type ClC‐6. Finally, rClC‐7R760Q, homologous to hClC‐7R762Q, an osteopetrosis variant with fast gating kinetics, appeared indifferent to extracellular Cl−, identifying altered Cl− sensitivity as a plausible mechanism underlying disease. Collectively, the present studies underscore the distinct roles of ClC‐6 and ClC‐7 within the context of their respective localization to late endosomes and lysosomes. In particular, we suggest the atypical inhibition of ClC‐7 by luminal Cl− serves to limit excessive intraluminal Cl− accumulation. Key points: ClC‐6 and ClC‐7 are late endosomal and lysosomal 2 Cl−/1 H+ exchangers, respectively. When targeted to the plasma membrane, both activate slowly at positive voltages.ClC‐6 activity is decreased in low extracellular (i.e. luminal) chloride, whereas ClC‐7 is activated by low luminal chloride, even at acidic pH.The functional gain‐of‐function phenotypes of the ClC‐6 and ClC‐7 disease mutations ClC‐6Y553C and ClC‐7Y715C are maintained when introduced in their respective homologues, ClC‐7Y577C and ClC‐6Y781C, with all mutations retaining chloride dependence of the respective wild type (WT).An osteopetrosis mutation of ClC‐7 displaying fast gating kinetics (R762Q) was less sensitive to extracellular chloride compared to WT.The opposing substrate dependences of ClC‐6 and ClC‐7 Cl− / H+ exchangers point to non‐overlapping physiological functions, leading us to propose that inhibition of ClC‐7 by luminal chloride and protons serves to prevent osmotic stress imposed by hyper‐accumulation of chloride. [ABSTRACT FROM AUTHOR]

Endosomes and lysosomes are intracellular vesicular organelles with important roles in cell functions such as protein homeostasis, clearance of extracellular material, and autophagy. Endolysosomes are characterized by an acidic luminal pH that is critical for proper function. Five members of the gene family of voltage-gated ChLoride Channels (CLC proteins) are localized to endolysosomal membranes, carrying out anion/proton exchange activity and thereby regulating pH and chloride concentration. Mutations in these vesicular CLCs cause global developmental delay, intellectual disability, various psychiatric conditions, lysosomal storage diseases, and neurodegeneration, resulting in severe pathologies or even death. Currently, there is no cure for any of these diseases. Here, we review the various diseases in which these proteins are involved and discuss the peculiar biophysical properties of the WT transporter and how these properties are altered in specific neurodegenerative and neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]