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 central nervous system, including hippocampal formation. Pathogenic variants recently found in these genes have given rise to the rare CLCN3- and CLCN4-neurodevelopmental conditions, characterised 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, as well as 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 hippocampus's Cornu Ammonis 2 (CA2) region: regular and burst firing. At post-natal day 13 (P13), 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 depolarising shift in the action potential threshold and an increase in the afterhyperpolarizing 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 arborisation. Branching and lengths of apical and basal domains were significantly reduced in the Clcn3-/- and moderately altered in the Clcn4-/- neurons. At P3, we found 25% of bursting neurons in Clcn3-/- with no significant morphological abnormalities in the dendritic arborisation compared to the wild-type, suggesting that functional defects precede structural changes in Cl-/H+ exchangers-deficient neurons. Similarly, 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 neurons' electrical excitability and firing patterns 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 toward potential therapeutic interventions for CLCN3- and CLCN4-related patients associated with disruption of Cl-/H+ exchange function.