Abstract
Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.
Highlights
Naϩ/Hϩ exchange is a process present in all organisms from prokaryotes to human [7]
IP3 receptorbinding protein released with IP3 (IRBIT) Interacts with Na؉/H؉ exchanger 3 (NHE3)—To identify proteins interacting with NHE3, we screened a human kidney cDNA library under high stringency with the GAL4 DNA-binding domain fused to the cytoplasmic domain of NHE3 as bait
Expression of IRBIT resulted in stimulation of NHE3 transport activity, whereas silencing of IRBIT inhibited NHE3 activity in response to increased Ca2ϩ
Summary
Plasmid Constructs—The bait plasmid, pGBKT7-C832, was constructed by cloning the coding region of the cytoplasmic COOH-terminal domain of rabbit NHE3 (aa 475– 832) into the yeast DNA-binding vector pGBKT7 vector (Clontech). Cells grown in 10-cm Petri dishes were treated with 100 nM thapsigargin, 500 nM ionomycin, or ethanol for 10 min, followed by rinsing twice in PBS and 10 min incubation in borate buffer composed of 154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl2, and 10 mM H3BO3 (pH 9.0). Confocal Immunofluorescence—PS120/NHE3V/HA-IRBIT and PS120/NHE3V/pcDNA cells grown on coverslips were washed twice with cold PBS, fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.2% Triton X-100 in PBS for 5 min, and blocked in PBS containing 5% normal goat serum for 30 min at room temperature. 10 min each, with PBS, the cells were incubated with Alexa 488-conjugated donkey anti-mouse IgG or Alexa 555-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature. Statistical significance was assessed by analysis of variance, with p Ͻ 0.05 considered significant
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