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trafficking. Methods: In the present study, we employed fluorescence resonance energy transfer (FRET) and Zeiss 510 confocal microscopy to study direct binding of NHE3 and NHERF2 in living or fixed polarized kidney epithelial cells (OK cells) that were transiently transfected with CFP-NHERF2 and YFP-NHE3. Results: Under basal conditions, NHERF2 and NHE3 exhibited 10-20% FRET signaling solely at the microvilli (an average of 10 regions of interest (ROIs) in the microvilli of each cells) but not at the juxtanuclear region. As a negative control, there was no FRET signaling with expression of YFP-GPI and CFP-NHERF2. Further, treatment with 10 μMCa2+ ionophore, which increases intracellular Ca2+, abolished the NHERF2/NHE3 FRET signaling within a minute. To further elucidate the role of Ca2+ signaling in regulation of NHE3 trafficking, we treated Caco-2/HA-NHE3 cells with carbachol (10 uM) that activates M3 muscarinic receptors and increases intracellular Ca2+ and monitored its effect on the trafficking of NHE3 via total internal reflection fluorescence (TIRF) microscopy. Carbachol reduced the surface level of NHE3 with an onset within one minute. Conclusions: 1) Under basal conditions, NHE3 binds NHERF2 in the OK cell microvilli and this binding can be quantified by acceptor photobleaching FRET. 2) Within one minute of elevation of intracellular Ca2+, the FRET signal between NHE3 and NHERF2 is abolished in OK cells. 3) Carbachol causes a similarly rapid reduction of surface NHE3 observed via TIRF. We conclude that elevation of intracellular Ca2+ leads to dissociation of NHERF2 from NHE3 at the microvillus which allows NHE3 transport activity to be inhibited by facilitating endocytosis. |
