Abstract
Objective. The study’s objective was to determine amino acids important for activation of human ClC‐2 (hClC‐2) by lubiprostone. NMR studies of ClC‐0 by Alioth et al (J Mol Biol 369:1163‐9, 2007) identified an α‐helical region that interacts with ClC‐0 CBS domains, important to channel regulation, which contains one of two PKA phosphorylation sites unique to hClC‐2 (RGET691).Methods. Whole cell patch clamp of wild type and mutant hClC‐2 expressed in EBNA293 cells were carried out as described (Cuppoletti et al, Cell Biochem Biophys 66: 53‐63, 2013). Wild type rabbit and mouse ClC‐2 were also studied.Results. Single amino acid mutagenesis of the PKA phosphorylation site RGET691‐>A shifted the EC50 for lubiprostone stimulation of wild type hClC‐2 from 27.6 ± 4.5 nM (4) to 445.8 ± 2.1 nM (3). The triple mutant RGET691‐>AGAA lost all lubiprostone stimulation. Both mutants were still stimulated by forskolin/IBMX. Both rabbit and mouse ClC‐2 were also stimulated by lubiprostone with EC50s of 350 ± 37.9 nM (3) and 64.3 ± 3.7 nM (4), respectively.Conclusions. RGET691 plays an essential role in lubiprostone stimulation of human ClC‐2. In light of NMR evidence that this region may interact with CBS domains and contains the RGET691 phosphorylation site in hClC‐2, binding of lubiprostone to this region in human, rabbit and mouse ClC‐2 may alter the interaction of this region with CBS domains, leading to direct modulation of ClC‐2.Grant Funding Source: Supported by Sucampo AG
Published Version
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