Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts
ABSTRACT The Cl/HCO(3) exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.
- SourceAvailable from: Jaleh Malakooti[Show abstract] [Hide abstract]
ABSTRACT: The epithelial apical membrane Na+/H+ exchangers [NHE (sodium hydrogen exchanger)2 and NHE3] and Cl-/HCO3- exchangers [DRA (down-regulated in adenoma) and PAT-1 (putative anion transporter 1)] are key luminal membrane transporters involved in electroneutral NaCl absorption in the mammalian intestine. During the last decade, there has been a surge of studies focusing on the short-term regulation of these electrolyte transporters, particularly for NHE3 regulation. However, the long-term regulation of the electrolyte transporters, involving transcriptional mechanisms and transcription factors that govern their basal regulation or dysregulation in diseased states, has only now started to unfold with the cloning and characterization of their gene promoters. The present review provides a detailed analysis of the core promoters of NHE2, NHE3, DRA and PAT-1 and outlines the transcription factors involved in their basal regulation as well as in response to both physiological (butyrate, protein kinases and probiotics) and pathophysiological (cytokines and high levels of serotonin) stimuli. The information available on the transcriptional regulation of the recently identified NHE8 isoform is also highlighted. Therefore the present review bridges a gap in our knowledge of the transcriptional mechanisms underlying the alterations in the gene expression of intestinal epithelial luminal membrane Na+ and Cl- transporters involved in electroneutral NaCl absorption. An understanding of the mechanisms of the modulation of gene expression of these transporters is important for a better assessment of the pathophysiology of diarrhoea associated with inflammatory and infectious diseases and may aid in designing better management protocols.Biochemical Journal 04/2011; 435(2):313-25. DOI:10.1042/BJ20102062 · 4.78 Impact Factor
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ABSTRACT: SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.AJP Cell Physiology 12/2011; 302(5):C781-95. DOI:10.1152/ajpcell.00165.2011 · 3.67 Impact Factor
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ABSTRACT: Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl(-) and HCO(3)(-) are essential for capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm capacitation, the intracellular concentration of Cl(-) ([Cl(-)](i)) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl(-) equilibrium potential appears to be close to the cell resting Em, opening of Cl(-) channels could not support the [Cl(-)](i) increase observed during capacitation. Alternatively, the [Cl(-)](i) increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl(-)](i) and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na(+)/H(+) regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytochemistry, Western blot, and immunoprecipitation data indicate that SLC26A3, SLC26A6, and SLC9A3R1 are expressed in mouse sperm, localize to the midpiece, and interact between each other and with CFTR. Moreover, we present evidence indicating that CFTR and SLC26A3 are involved in the [Cl(-)](i) increase induced by db-cAMP in noncapacitated sperm. Furthermore, we found that inhibitors of SLC26A3 (Tenidap and 5099) interfere with the Em changes that accompany capacitation. Together, these findings indicate that a CFTR/SLC26A3 functional interaction is important for mouse sperm capacitation.Biology of Reproduction 01/2012; 86(1):1-14. DOI:10.1095/biolreprod.111.094037 · 3.45 Impact Factor