Mutations in the CLCN5 (chloride channel, voltage-sensitive 5) gene cause Dent's disease because they reduce the functional expression of the ClC-5 chloride/proton transporter in the recycling endosomes of proximal tubule epithelial cells. The majority (60%) of these disease-causing mutations in ClC-5 are misprocessed and retained in the ER (endoplasmic reticulum). Importantly, the structural basis for misprocessing and the cellular destiny of such ClC-5 mutants have yet to be defined. A ClC-5 monomer comprises a short N-terminal region, an extensive membrane domain and a large C-terminal domain. The recent crystal structure of a eukaryotic ClC (chloride channel) transporter revealed the intimate interaction between the membrane domain and the C-terminal region. Therefore we hypothesized that intramolecular interactions may be perturbed in certain mutants. In the present study we examined two misprocessed mutants: C221R located in the membrane domain and R718X, which truncates the C-terminal domain. Both mutants exhibited enhanced protease susceptibility relative to the normal protein in limited proteolysis studies, providing direct evidence that they are misfolded. Interestingly, the membrane-localized mutation C221R led to enhanced protease susceptibility of the cytosolic N-terminal region, and the C-terminal truncation mutation R718X led to enhanced protease susceptibility of both the cytosolic C-terminal and the membrane domain. Together, these studies support the idea that certain misprocessing mutations alter intramolecular interactions within the full-length ClC-5 protein. Further, we found that these misfolded mutants are polyubiquitinated and targeted for proteasomal degradation in the OK (opossum kidney) renal epithelial cells, thereby ensuring that they do not elicit the unfolded protein response.

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