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Background/Aims: Enterocytes express a number of NHE isoforms with presumed localization in the apical (NHE2, 3 and 8) or basolateral (NHE1) membrane. Functional activity and localization of enterocyte NHE isoforms were assessed using fully differentiated Caco-2BBe cells, whose genetic expression profile closely resembles mature enterocytes. Methods: The activity of the different NHEs was analyzed by fluorometric pHi-metry in a perfusion chamber with separate apical and basolateral perfusion, using specific inhibitors and shRNA knockdown of NHE2. The expression of the NHEs and of other relevant acid extrusion transporters was quantified by qPCR. Results: Quantitative comparison of the mRNA expression levels of the different NHE isoforms in 14 day-differentiated Caco-2BBe cells showed the following order: NHE2>NHE8>NHE3>NHE1. Acid-activated NHE exchange rates in the basolateral membrane were >6-fold higher than in the apical membrane. 79 ± 3 % of the acid-activated basolateral Na+/H+ exchange rate displayed a NHE1-typical inhibitor profile, and no NHE2/3/8 typical activity could be observed. Analysis of the apical Na+/H+ exchange rates revealed that approximately 51 ± 3 % of the total apical activity displayed a NHE2/8-typical inhibitor profile and 31 ± 6 % a NHE3-typical inhibitor profile. Because no selective NHE2 inhibitor is available, a stable NHE2 knockdown cell line (C2NHE2KD) was generated. C2NHE2KD displayed a reduced NHE2-typical apical Na+/H+ exchange rate and maintained a lower steady-state pHi, despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7). Conclusion: Differentiated Caco-2BBe cells display particularly high mRNA expression levels of NHE2, which can be functionally identified in the apical membrane. Although at low intracellular pH, NHE2 transport rate was far lower than that of NHE1. NHE2 activity was nevertheless essential for the maintenance of the steady-state pHi of these cells.

Background/Aims: The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3– movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. Methods: Results: In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Conclusion: Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed.

The epithelial Ca2+ channel TRPV5 (ECaC1) plays a key role in renal and intestinal Ca2+ (re)absorption and is thus regulated by 1,25(OH) 2D3. The present study aims to explore whether TRPV5 is regulated by the serum and glucocorticoid inducible kinase SGK1, a kinase transcriptionally upregulated by 1,25(OH) 2D3. To this end cRNA encoding TRPV5 has been injected into Xenopus oocytes with or without additional injection of SGK1, its isoforms SGK2 and SGK3, constitutively active (S422D)SGK1, inactive (K127N)SGK1, constitutively active (T308D,S473D)PKB and/or the Na+/H+ exchanger regulating factor NHERF2. In Xenopus laevisoocytes expression of TRPV5 increases uptake of tracer Ca(S422D;) and induces a Ca2+ current (ICa). In the presence of Cl-, TRPV5 mediated Ca2+ entry leads to secondary activation of Ca(2+)-sensitive Cl- channels (ICl(Ca)). Coexpression of TRPV5 with both (S422D)SGK1 and NHERF2 stimulates tracer Ca2+ entry, ICa and ICl(Ca). The effect of (S422D)SGK1 on TRPV5 and NHERF2 expressing oocytes is mimicked by SGK1 and SGK3, but not by SGK2, constitutively active (T308D,S473D)PKB or inactive (K127N)SGK1. The observations suggest that SGK1, SGK3 and NHERF2 regulate TRPV5 and are thus likely to participate in the regulation of calcium homeostasis.

Na+/H+ exchanger regulatory factors, NHERF1 and NHERF2, are structurally related proteins and highly expressed in epithelial cells. These proteins are initially identified as accessory proteins in the regulation of Na+/H+ exchanger isoform 3, NHE3. In addition to regulation of NHE3, recent studies demonstrate the importance of NHERF1 and NHERF2 in recycling and localization of membrane receptors, ion channels and transporters. Recent studies show that serum- and glucocorticoid-induced kinase 1 (SGK1) specifically interacts with NHERF2 but not with NHERF1, adding to the growing number of differences between the two proteins. The association of SGK1 with NHERF2 is necessary for stimulation of NHE3 activity by glucocorticoids. In addition, SGK1 together with NHERF2 stimulates the K+ channel ROMK1, suggesting a broader role of SGK1 in regulation of ion transport.

Na+/H+exchange was first described by Murer, Hopfer and Kinne [19] in renal brush border membrane vesicles. This process is mediated by Na+/H+exchangers which catalyze the exchange of extracellular Na+for intracellular H with a stoichiometry of 1:1. Na+/H+exchangers have multiple functions, including pH homeostasis, volume regulation, cell proliferation, and transcellular Na+absorption [reviewed in 12]. In no cell is it the only mechanism for any one of these functions. For instance, multiple mechanisms of pH homeostasis are present in most eukaryotic cells including a c┌/HCO3-exchanger, a NaHCO3co-transporter, a Na+- dependent cr/HCO3-exchanger and multiple mechanisms of hT extrusion [reviewed in 15], including the H-K-ATPase pump. In this review, we will focus on recent advances in identification and understanding of the structure/function relationships and acute protein kinase regulation of members of the mammalian Na+/H+exchanger gene family.