Abstract
The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation.
Highlights
Introduction of autonomous calmodulindependent protein kinase II (CaMKII) into hCLC3 stably transfected tsA cells gave rise to an outwardly rectifying ClϪ current
We show that the plasma membrane expression of a member of the ClC family of Cl؊ channels, human CLC-3, a 90-kDa protein, is regulated by CaMKII
A single molecular mass band of ϳ90 kDa was obtained from the same whole cell lysates digested with N-glycanase, which was consistent with the predicted mass for human CLC-3 (hCLC-3) of 88 kDa
Summary
3 stably transfected tsA cells gave rise to an outwardly rectifying ClϪ current. The percentage of cells responsive to CaMKII (78%) was comparable to the positive immunostaining in the hCLC-3 stably transfected tsA cells (80%). The relative lyotropic anion permeability (PX/PCl) of wild type hClC-3 was IϪ Ͼ BrϪ Ͼ ClϪ and is consistent with previous reports of ICl,CaMKII from different laboratories (3, 16, 25–27). To confirm that ICl,CaMKII is directly mediated by hCLC-3 and not due to activation of an ion channel endogenous to tsA cells, a mutation in hCLC-3 (G280E) was made and transiently expressed in tsA cells. The relative anion permeability for the G280E mutant was changed to ClϪ Ͼ BrϪ Ͼ IϪ, supporting the hypothesis that the human isoform of CLC-3 codes for a CaMKII-activated conductance
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
