engleski

Calmodulin dependent protein kinase II (CaMKII) binds and inhibits NHE3 under basal conditions.

Introduction: Sodium/hydrogen exchangers (NHE) mediate counter transport of H+ for Na+ across cellular membranes, contributing to pH, volume regulation and transepithelial sodium transport. NHE3 is epithelial isoform highly expressed in kidney proximal tubules and intestinal cells where it regulates neutral sodium absorption. NHE3 regulation occurs during normal digestive processes and is found to be inhibited in diarrheal diseases. The ubiquitously expressed CaMKII is implicated in many aspects of cellular function. It is mainly studied in brain tissue, but our results show that CaMKII also participates in regulation of NHE3 activity. Aim: To show relationship between CaMKII and NHE3 using PS120 fibroblasts expressing rabbit NHE3. Methods: NHE3 activity was determined in PS120 cells transfected with both &plusmn ; NHERF2 (Na+/H+ exchanger regulatory factor) and either full length NHE3 or NHE3 truncation mutants. NHE3 activity was measured by calculating H+ efflux rate with spectrofluorimeter using BCECF (pH sensitive fluorescent dye) +/- KN62 (inhibitor of CaMKII). The association of CaMKII with wild NHE3 and NHE3 truncation mutants was tested by coimmunoprecipitation of PS120 lysates with anti-CaMKII antibodies. Binding was detected by immunoblotting. NHE truncation mutants (NHE/Δ 585, NHE/Δ 605, NHE/Δ 640) were constructed by PCR and transfected to PS120 cells. Results: CaMKII inhibited NHE3 activity under basal conditions and this inhibition is mediated by NHERF2, since KN62 stimulated activity by 25% only in the presence of NHERF2. CaMKII, in vivo, bound to NHE3 under basal conditions and its binding was calcium independent. The binding site was found to be within NHE3 cytoplasmic domain between a.a. 585 and 605. Conclusion: NHE3 is CaMKII binding protein. CaMKII binds to cytoplasmic part of NHE3 in calcium independent way. CaMKII regulate NHE3 under basal conditions and this regulation is mediated by NHERF2 associating protein.

NHE3; CaMKII; PS120; BCECF; KN62; Na+ absorption

nije evidentirano