Kir4.1 channel expression is essential for parietal cell control of acid secretion.

Kir4.1 channels were found to colocalize with the H(+)/K(+)-ATPase throughout the parietal cell (PC) acid secretory cycle. This study was undertaken to explore their functional role. Acid secretory rates, electrophysiological parameters, PC ultrastructure, and gene and protein expression were determined in gastric mucosae of 7-8-day-old Kir4.1-deficient mice and WT littermates. Kir4.1(-/-) mucosa secreted significantly more acid and initiated secretion significantly faster than WT mucosa. No change in PC number but a relative up-regulation of H(+)/K(+)-ATPase gene and protein expression (but not of other PC ion transporters) was observed. Electron microscopy revealed fully fused canalicular membranes and a lack of tubulovesicles in resting state Kir4.1(-/-) PCs, suggesting that Kir4.1 ablation may also interfere with tubulovesicle endocytosis. The role of this inward rectifier in the PC apical membrane may therefore be to balance between K(+) loss via KCNQ1/KCNE2 and K(+) reabsorption by the slow turnover of the H(+)/K(+)-ATPase, with consequences for K(+) reabsorption, inhibition of acid secretion, and membrane recycling. Our results demonstrate that Kir4.1 channels are involved in the control of acid secretion and suggest that they may also affect secretory membrane recycling.

The switch of intestinal Slc26 exchangers from anion absorptive to HCOFormula secretory mode is dependent on CFTR anion channel function.

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO(3)(-)) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO(3)(-) secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl(-)/HCO(3)(-) exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO(3)(-) secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO(3)(-) secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na(+)/H(+) exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl(-) absorptive mode and Cl(-) exit via CFTR to operate in the HCO(3)(-) secretory mode.

Duodenal acidity "sensing" but not epithelial HCO3- supply is critically dependent on carbonic anhydrase II expression.

Carbonic anhydrase (CA) is strongly expressed in the duodenum and has been implicated in a variety of physiological functions including enterocyte HCO(3)(-) supply for secretion and the "sensing" of luminal acid and CO(2). Here, we report the physiological role of the intracellular CAII isoform involvement in acid-, PGE(2,) and forskolin-induced murine duodenal bicarbonate secretion (DBS) in vivo. CAII-deficient and WT littermates were studied in vivo during isoflurane anesthesia. An approximate 10-mm segment of the proximal duodenum with intact blood supply was perfused under different experimental conditions and DBS was titrated by pH immediately. Two-photon confocal microscopy using the pH-sensitive dye SNARF-1F was used to assess duodenocyte pH(i) in vivo. After correction of systemic acidosis by infusion of isotonic Na(2)CO(3), basal DBS was not significantly different in CAII-deficient mice and WT littermates. The duodenal bicarbonate secretory response to acid was almost abolished in CAII-deficient mice, but normal to forskolin- or 16,16-dimethyl PGE(2) stimulation. The complete inhibition of tissue CAs by luminal methazolamide and i.v. acetazolamide completely blocked the response to acid, but did not significantly alter the response to forskolin. While duodenocytes acidified upon luminal perfusion with acid, no significant pH(i) change occurred in CAII-deficient duodenum in vivo. The results suggest that CA II is important for duodenocyte acidification by low luminal pH and for eliciting the acid-mediated HCO(3)(-) secretory response, but is not important in the generation of the secreted HCO(3)(-) ions.

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion.

Parietal cell (PC) proton secretion via H(+)/K(+)-ATPase requires apical K(+) recycling. A variety of K(+) channels and transporters are expressed in the PC and the molecular nature of the apical K(+) recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1(+/+), (+/-) and (-/-) mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1(-/-) and (+/+) mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by approximately 90% in KCNQ1(-/-) gastric mucosa. Application of a high K(+) concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1(-/-) mucosa. The study demonstrates that the KCNQ1 channel provides K(+) to the extracellular K(+) binding site of the H(+)/K(+)-ATPase during acid secretion, and no other gastric K(+) channel can substitute for this function.

Differential roles of NHERF1, NHERF2, and PDZK1 in regulating CFTR-mediated intestinal anion secretion in mice.

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na+/H+ exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3- secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3- secretory rates and blocked beta2-adrenergic receptor (beta2-AR) stimulation. Conversely, Nherf2-/- mice displayed augmented FSK-stimulated HCO3- secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3- secretion in WT mice, this effect was lost in Nherf2-/- mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3- secretion. In addition, laser microdissection and quantitative PCR revealed that the beta2-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the beta2-AR and CFTR was reduced in the Nherf1-/- mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3- secretion: while NHERF1 is an obligatory linker for beta2-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

Localization, trafficking, and significance for acid secretion of parietal cell Kir4.1 and KCNQ1 K+ channels.

BACKGROUND & AIMS: K(+) recycling at the apical membrane of gastric parietal cells is a prerequisite for gastric acid secretion. Two K(+) channels are currently being considered for this function, namely KCNQ1 and inwardly rectifying K(+) channels (Kir). This study addresses the subcellular localization, trafficking, and potential functional significance of KCNQ1 and Kir4.1 channels during stimulated acid secretion. METHODS: The effect of pharmacologic KCNQ1 blockade on acid secretion was studied in cultured rat and rabbit parietal cells and in isolated mouse gastric mucosa. The subcellular localization of KCNQ1 and Kir4.1 was determined in highly purified membrane fractions by Western blot analysis as well as in fixed and living cells by confocal microscopy. RESULTS: In cultured parietal cells and in isolated gastric mucosa, a robust acid secretory response was seen after complete pharmacologic blockade of KCNQ1. Both biochemical and morphologic data demonstrate that Kir4.1 and KCNQ1 colocalize with the H(+)/K(+)-ATPase but do so in different tubulovesicular pools. All Kir4.1 translocates to the apical membrane after stimulation in contrast to only a fraction of KCNQ1, which mostly remains cytoplasmic. CONCLUSIONS: Acid secretion can be stimulated after complete pharmacologic blockade of KCNQ1 activity, suggesting that additional apical K(+) channels regulate gastric acid secretion. The close association of Kir4.1 channels with H(+)/K(+)-ATPase in the resting and stimulated membrane suggests a possible role for Kir4.1 channels during the acid secretory cycle.

Sodium and chloride absorptive defects in the small intestine in Slc26a6 null mice.

PAT1 (Slc26a6) is located on the apical membrane of the small intestinal villi, but its role for salt absorption has not been studied. To ascertain the role of Slc26a6 in jejunal sodium and chloride absorption, and its interplay with NHE3, muscle-stripped jejuna from Slc26a6+/+ and -/- and NHE3 +/+ and -/- mice were mounted in Ussing chambers and electrical parameters, and (36)Cl(-) and (22)Na(+) fluxes were measured. In parallel studies, expression of the apical Na(+)/H(+) exchanger (NHE3) was examined by immunofluorescence labeling and immunoblot analysis in brush border membrane (BBM). In the basal state, net Cl(-) and Na(+) fluxes were absorptive in Slc26a6-/- and +/+ jejuni, but significantly decreased in -/- animals. Upon forskolin addition, net Na(+) absorption decreased, Isc strongly increased, and net Cl(-) flux became secretory in Slc26a6-/- and +/+ jejuni. When luminal glucose was added to activate Na(+)/glucose cotransport, concomitant Cl(-) absorption was significantly reduced in Slc26a6 -/- jejuni, while Na(+) absorption increased to the same degree in Slc26a6 -/- and +/+ jejuni. Identical experiments in NHE3-deficient jejuni also showed reduced Na(+) and Cl(-) absorption. Results further demonstrated that the lack of NHE3 rendered Na(+) and Cl(-) absorption unresponsive to inhibition by cAMP, but did not affect glucose-driven Na(+) and Cl(-) absorption. Immunoblotting revealed comparable NHE3 abundance and distribution in apical membranes in Slc26a6-/- and +/+ mice. The data strongly suggests that Slc26a6 acts in concert with NHE3 in electroneutral salt absorption in the small intestine. Slc26a6 also serves to absorb Cl(-) during glucose-driven salt absorption.