Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume

Abstract

Regulation of cell volume is essential for every cell and is accomplished by the regulated loss or gain of intracellular ions or other osmolytes. Regulatory volume decrease often involves the parallel activation of potassium and chloride channels. Overexpression of P-glycoprotein leads to volume-activated Cl- currents but its physiological importance for volume regulation is unclear. CIC-2 is a ubiquitously expressed Cl- channel activatable by non-physiologically strong hyperpolarization. We now show that CIC-2 can be activated by extracellular hypotonicity, which suggests that it has a widespread role in volume regulation. Domains necessary for activation by both voltage and volume are localized to the amino terminus. Mutations in an 'essential' region lead to constitutively open channels unresponsive to medium tonicity, whereas deletions in a 'modulating' region produce partially opened channels responsive to both hypo- and hypertonicity. These domains can be transplanted to different regions of the protein without loss of function.

Evidence for a Functional Interaction between the ClC-2 Chloride Channel and the Retrograde Motor Dynein Complex

“…In our future work, we plan to determine the role of dynein-mediated vesicular trafficking in ClC-2 activation by experimental maneuvers shown previously to regulate ClC-2 function at the cell surface. For example, activation of protein kinase C, cyclin-dependent kinase p34 cdc2 /cyclin B, and phosphatidylinositol 3-kinase have been shown to inhibit ClC-2 function at the cell surface (12,32,42,43). Furthermore, Zheng et al (43) showed that cyclin B-dependent phosphorylation of rabbit ClC-2 led to its enhanced ubiquitination and degradation.…”

“…As both antibodies are effective in immunoprecipitating similar amounts of protein, this result possibly implicates the N terminus of ClC-2 in mediating its interaction with the dynein complex. The N terminus of ClC-2 has been implicated previously in the regulation of the channel function of the protein (32). However, our preliminary in vitro binding experiments (including GST-fusion pull-down and enzyme-linked immunosorbent assay assays) failed to provide evidence supporting a specific, direct interaction between purified dynein (kindly provided by S. King and T. Schroer) and GST-fusion proteins containing ei- ther the N terminus (residues 31-74) or C terminus (residues 869 -907) of ClC-2.…”

Additional Disruption of the ClC-2 Cl- Channel Does Not Exacerbate the Cystic Fibrosis Phenotype of Cystic Fibrosis Transmembrane Conductance Regulator Mouse Models

“…ClC-2 is activated by hyperpolarization, cell swelling, and acidic extracellular pH (15,20,23). Many physiological functions have been postulated for ClC-2 (9), but blindness and male infertility were, surprisingly, the only phenotypes noted in ClC-2 knock-out mouse models (24,25).…”

Molecular cloning and characterization of a novel truncated form (ClC‐2β) of ClC‐2α (ClC‐2G) in rabbit heart

“…However, without sequence analysis and knowledge of intron/exon organization of the gene, it is not known whether rabC1C-2a and -2fl are alternative splicing products of the common initial transcripts or whether they are products derived from distinct alternative promoter regions. Expressed rC1C-2 current was enhanced when oocytes were swollen by superfusion with hypotonic solution [27]. Deletion mutation from 5'-end of rC1C-2 loses sensitivity to cell swelling, resulting in an open channel even in the absence of cell swelling [27].…”

“…Expressed rC1C-2 current was enhanced when oocytes were swollen by superfusion with hypotonic solution [27]. Deletion mutation from 5'-end of rC1C-2 loses sensitivity to cell swelling, resulting in an open channel even in the absence of cell swelling [27]. Thus, 5'-region of rC1C-2 was suggested as a critical site for determination of sensitivity to cell swelling [25].…”