A serine residue in ClC-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume.
In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.
Duan
D
D
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046, USA.
Cowley
S
S
Horowitz
B
B
Hume
J R
JR
D000818:Animals; D002460:Cell Line; D048429:Cell Size; D018118:Chloride Channels / Q000378:metabolism*; D004558:Electric Stimulation; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D008564:Membrane Potentials / Q000502:physiology; D009206:Myocardium / Q000166:cytology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012694:Serine / Q000378:metabolism*
D018118:Chloride Channels; D004791:Enzyme Inhibitors; D012694:Serine; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases