CLCN4 Membrane Protein Introduction

Introduction of CLCN4

H(+)/Cl(-) exchange transporter 4 (CLCN4), also known as chloride channel protein 4, is one of the nine members of CLCN family. It has an evolutionary conserved CpG island and is conserved in both mouse and hamster. This gene is mapped in close proximity to APXL (Apical protein Xenopus laevis-like) and OA1 (Ocular albinism type I), which are both located on the human X chromosome. The physiological role of chloride channel 4 remains unclear but may contribute to the pathogenesis of neuronal disorders. Alternate splicing shows us two transcript variants that encode different proteins isoform.

Function of CLCN4 Membrane Protein

The CLC channel family contains members including chloride channels and proton-coupled anion transporters which can exchange chloride or another anion for protons. The presence of conserved gating glutamate residues is tightly related to function as antiporters for family members. As a vital member of CLC channel family, H(+)/Cl(-) exchange transporter 4 always plays the role as proton-coupled chloride transporter, antiport system and exchanges chloride ions against protons. Each site has its own work. For example, the site at the position of 224 mediates proton transfer from the outer aqueous phase to the interior of the protein, as well as transportation of H(+) and Cl(-). The binding site at the position of 610 takes part in activity with ATP via amide nitrogen and carbonyl oxygen. In conclusion, CLCN4 involves in antiporter activity, ATP binding, chloride channel activity and voltage-gated chloride channel activity. Activation of the CLCN4 can participate in sensations such as pain, warmth, cold, taste pressure and vision through stimuli-sensing channels.

Fig1. Apparently pathogenic CLCN4 mutations identified in the screen and functional analysis of the missense variants. (Hu, 2015)

Application of CLCN4 Membrane Protein in Literature

1. Veeramah K.R., et al. Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. Epilepsia. 2013, 54 (7): 1270-81. PubMed ID: 23647072
   
   

   The article identified more than one de novo variants that are predicted to alter protein function with a percentage of 90. The de novo variants are in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The results of in vitro analyses using cell-based assays reveal that the CLCN4 mutation greatly impairs ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the mutation in ARHGEF15 reduces GEF exchange activity of the gene product, Ephexin5, by about 50%.

2. Picollo A., et al. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature. 2005, 436(7049):420-3. PubMed ID: 16034421
   
   

   This article finds that when ClC-4 and ClC-5 are activated by positive voltages, they can carry a substantial amount of protons across the plasma membrane, which can be detected by measurements of pH close to the cell surface. Both of them are able to extrude protons against their electrochemical gradient, demonstrating secondary active transport. Cl^- and H⁺ transportation contributes equally to the total charge movement in CIC-5, which results in a raising possibility that the coupled Cl^-/H⁺ transport of ClC-4 and ClC-5 is of significant magnitude in vivo.

3. Okkenhaug H., et al. The human ClC-4 protein, a member of the CLC chloride channel/transporter family, is localized to the endoplasmic reticulum by its N-terminus. FEBS J. 2005, 272 (19): 4996–5007. PubMed ID: 16176272
   
   

   This article suggests that differences between the structures of CLIC1 and CLIC4 are resulted from helix 2 in the glutaredoxin- like N-terminal domain. This difference has previously been reported to undergo a dramatic structural change in CLIC1 upon oxidation. Membrane binding is enhanced by oxidation of CLIC4. However, no channels are observed via tip-dip electrophysiology with the presence of a reducing agent. Recombinant CLIC4 seems to be able to form a redox-regulated ion channel in the condition of absence of any other proteins.

4. Huang L., et al. Identification and functional characterization of a voltage-gated chloride channel and its novel splice variant in taste bud cells. J Biol Chem. 2005, 280(43):36150-7. PubMed ID: 16129671
   
   

   This article reports the molecular identification and functional characterization of a voltage-gated chloride channel (ClC-4) and its novel splice variant (ClC-4A) from taste bud cells. Their results show that ClC-4A is a candidate channel for an acid-induced 5-nitro-2-(3-phenylpropylamino) benzoic acid-sensitive current. What’s more, these two channels may play a role in bitter-, sweet-, and umami-mediated taste transmission by regulating transmitter uptake into synaptic vesicles.

5. Palmer E.E., et al. De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females. Mol Psychiatry. 2018, 23(2):222-230. PubMed ID: 27550844
   
   

   In this article, all missense variants are predicted to have effect on CLCN4's function based on in silico tools and either segregated with the phenotype in the family or are de novo. Pathogenicity of all previously unreported missense variants is further supported by electrophysiological studies in Xenopus laevis oocytes. Authors also compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.

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Reference

1. Hu H, et al. (2015). X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes. Molecular Psychiatry. 21(1), 133-48.

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