Functional Characterization of CLCN4 Variants Associated With X-Linked Intellectual Disability and Epilepsy

Abstract

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

“…Their physiological importance has been demonstrated by studies of knockout animal models and by naturally occurring mutations in patients with genetic diseases: genetic ablation or naturally occurring mutations in CLCN3 or CLCN7 causes neurodegeneration in the central nervous system (CNS; Kornak et al, 2001 ; Stobrawa et al, 2001 ; Dickerson et al, 2002 ; Kasper et al, 2005 ; Duncan et al, 2021 ). Mutations in the CLCN4 gene are associated with intellectual disability and epilepsy ( Veeramah et al, 2013 ; Hu et al, 2016 ; Palmer et al, 2018 ; He et al, 2021b ; Guzman et al, 2022 ), and CLCN6 mutations result in West syndrome and lysosomal storage disease ( Poet et al, 2006 ; He et al, 2021a ). Genetic ablation of ClC-6 or downregulation of ClC-3 alters pain sensitivity in mice ( Poet et al, 2006 ; Pang et al, 2016 ), suggesting a role for Cl – /H + exchangers in pain regulation.…”

Determination of oligomeric states of proteins via dual-color colocalization with single molecule localization microscopy

“…Independence of both fluorescence signals was guaranteed by using proteins with separate emission and excitation spectra as well as performing sequential rather than simultaneous imaging of color channels. However, based on our experience with solubilizing ion channels and transporters tagged with fluorescent proteins ( Guzman et al, 2022 ; Stölting et al, 2014 ; Stölting et al, 2015a ; Tan et al, 2017 ), we expected that a fraction of fluorescent proteins might be cleaved off within the linker region or within M, leading to the simultaneous removal of both F and M. We introduced an additional factor, m , to describe the probability of this phenomenon occurring. Notwithstanding that we cannot exclude other possibilities contributing to the loss of both fluorescent proteins, m remains generally as the probability of both M and F simultaneously being dysfunctional.…”

Determination of oligomeric states of proteins via dual-color colocalization with single molecule localization microscopy

“…Independence of both fluorescence signals was guaranteed by using proteins with separate emission and excitation spectra as well as performing sequential rather than simultaneous imaging of color channels. However, based on our experience with solubilizing ion channels and transporters tagged with fluorescent proteins (Guzman et al, 2022; Stölting et al, 2014; Stölting, Bungert-Plümke, et al, 2015; Tan et al, 2017), we expected that a small fraction of fluorescent proteins might be cleaved off within the linker region or within M, leading to the simultaneous removal of both F and M. We introduced an additional factor, m , to describe the probability of this phenomenon occurring. Notwithstanding that we cannot exclude other possibilities contributing to the loss of both fluorescent proteins, m remains generally as the probability of both M and F simultaneously being dysfunctional.…”