Endosomal 2Cl−/H+ exchangers regulate neuronal excitability by tuning Kv7/KCNQ channel density

CLCN3 and CLCN4 encode the endosomal 2Cl-/H+ exchangers ClC-3 and ClC-4, which are highly expressed within the CNS, including the hippocampal formation. Pathogenic variants recently found in these genes have given rise to the rare CLCN3- and CLCN4-related neurodevelopmental conditions, characterized by a range of neurological and neuropsychiatric complications, such as global developmental delay, intellectual disability as a core feature, seizures, behavioural issues and brain abnormalities. The mechanisms by which ClC-3 and ClC-4 regulate neuronal function and viability, in addition to the molecular pathways affected in CLCN3- and CLCN4-related neurodevelopmental conditions, remain unknown. In neurodegenerative diseases, neuronal dendrites undergo pathological changes often associated with aberrant electrical activity. To investigate how ClC-3 or ClC-4 deficit alters neuronal excitability and morphology, we combined patch-clamp recordings in acute hippocampal slice preparations with simultaneous intracellular biocytin filling. We analysed the functional and structural properties of Clcn3-/- and Clcn4-/- neurons. Two firing patterns are found in the cornu ammonis 2 (CA2) region of the hippocampus: regular and burst firing. At postnatal Day 13, 62% of the assessed CA2 wild-type neurons showed a rhythmic bursting behaviour; this was reduced to 19% in Clcn4-/- and completely absent in the Clcn3-/- condition. Changes in the firing patterns were accompanied by a depolarizing shift in the action potential threshold and an increase in the after-hyperpolarizing phase of the action potentials. Blockade of Kv7/KCNQ and, to a lesser extent, Kv1, but not BK, SK or Kv2 channels, recapitulates the wild-type firing pattern phenotype in the Clcn3-/- condition. Moreover, we detected abnormalities in the complexity of the dendritic arborization. Branching and lengths of apical and basal domains were significantly reduced in the Clcn3-/- neurons and moderately altered in the Clcn4-/- neurons. At postnatal Day 3, we found 25% of bursting neurons in Clcn3-/- with no significant morphological abnormalities in the dendritic arborization in comparison to the wild-type, suggesting that functional defects precede structural changes in Cl-/H+ exchanger-deficient neurons. Likewise, dentate granule cells exhibited defective action potential properties and reduced burst-firing activity, which was substantially but not fully rescued by Kv7/KCNQ blockage. We conclude that Cl-/H+ exchangers regulate the electrical excitability and firing patterns of neurons primarily by fine-tuning Kv7/KCNQ channel density, and that functional defects might contribute to alterations in dendritic morphology. Our findings provide new insights into the underlying molecular mechanisms of Cl-/H+ exchangers in neurons and pave the way for potential therapeutic interventions for CLCN3- and CLCN4-related patients associated with disruption of Cl-/H+ exchange function.