Unravelling a role for KCNQ1 in K+ recycling and gastric acid secretion.

Gastric acid secretion by the luminal H+/K+-ATPase is dependent on recycling of K+ from the cell to the lumen to supply K+ for the H+–K+ exchange mechanism. Several different K+ channels, depending on the species studied, have been suggested to support gastric acid secretion, including KCNQ1 and the inward rectifying channels Kir1.1, Kir2.1, Kir4.1 and Kir5.1. KCNQ1 has been speculated to be the primary recycling K+ channel in gastric parietal cells for almost a decade, and several lines of evidence point to a pivotal role of KCNQ1 in gastric acid secretion. KCNQ1 is a member of a large family of voltage-activated K+ channels and regulates key physiological functions such as shaping the cardiac action potential and controlling water and salt homeostasis in several epithelial tissues. As such, it has been shown to be abundantly expressed in stomach, small and large intestine, kidneys and pancreas. KCNQ1 and the regulatory subunit KCNE2 are highly expressed and co-localize in gastric parietal cells. The assembly of KCNQ1 with KCNE2 in the apical membrane of the parietal cell is of special importance considering the extreme condition of pH 1 at the luminal side. KCNE2 changes the biophysical properties of KCNQ1 and confers activation by external acidification on KCNQ1. This is essential because KCNQ1 alone is inhibited by low extracellular pH and would not be able to conduct K+ under such conditions. These data suggest KCNE2 plays a critical role as the regulatory subunit of KCNQ1 in parietal cells; indeed, KCNE2-deficient mice exhibit severely impaired gastric acid secretion as well as disturbed gastric morphology. The first evidence for a crucial role of a distinct K+ channel in gastric acid secretion was provided by Lee et al. (2000), who observed an unexpected gastric phenotype in KCNQ1 knockout (KCNQ1−/−) mice. They found that KCNQ1−/− mice exhibited severe hyperplasia of the antrum and fundus mucosa, disturbed organization of the middle portion of the gastric glands, and a decreased number of parietal cells. Pharmacological inhibition of KCNQ1 by the chromanol 293B (a selective KCNQ1 blocker) in vitro and in vivo also resulted in decreased gastric acid secretion in mice, rats, rabbits and dogs (for review see Heitzmann & Warth, 2008). Further, it was shown by Seidler's group that cell shrinkage, which occurs after stimulation of acid secretion in gastric rabbit parietal cells, is dependent on K+ and is sensitive to the chromanol 293B (Bachmann et al. 2007). Elegant studies by Vallon et al. (2005) demonstrated that the recovery rate to neutral pH after an NH4+ pulse was almost completely absent in KCNQ1−/− mice compared to wild-type (WT) mice that were prestimulated with histamine or carbachol. Taken together, these results indicate on the cellular level an important role for KCNQ1 in gastric acid secretion. Although studies using adult KCNQ1−/− mice demonstrate impaired gastric acid secretion, the interpretation of the loss of acid secretion is complicated due to their complex gastric phenotype. A recent report by Song et al. (2009) in The Journal of Physiology provided new insight using an excellent approach: they studied the acid secretory capacity of gastric mucosa in very young KCNQ1−/− mice before they developed severe changes in gastric morphology. Of note, it is technically challenging to use mice at an age between 7–8 days for the performed experiments, particularly Ussing chamber measurements. Despite this, the authors found that the gastric mucosa of 7- to 8−day-old KCNQ1−/− mice did not exhibit any spontaneous acid secretion. Along those lines, KCNQ1−/− mice had a significantly higher gastric mucosal transepithelial resistance (Rt) compared to WT mice under resting conditions, which is most probably attributed to a reduced apical membrane conductance. Further, stimulated KCNQ1−/− gastric mucosa revealed a significant reduction in forskolin-induced acid secretion compared to WT, and although forskolin-stimulation reduced Rt in both genotypes, Rt remained significantly higher in KCNQ1−/− mucosa. Interestingly, forskolin stimulation resulted in a significantly greater increase in Isc and potential difference in KCNQ1−/− compared to WT mice. The authors suggest that this increase reflects the electrogenic part of agonist-stimulated anion (Cl−) secretion. This is in contrast to findings in adult KCNQ1−/− mice which exhibited a 50% reduction in forskolin-stimulated jejunal Cl− secretion (Vallon et al. 2005), and in rabbit and rat colon where pharmacological blockade of KCNQ1 almost completely prevented forskolin-induced Cl− secretion (Lohrmann et al. 1995). It would be helpful to perform experiments, such as 37Cl−-flux studies, using gastric mucosa from young KCNQ1−/− and WT mice to determine if there is a direct or indirect role of KCNQ1 for Cl− secretion. To test the hypothesis that KCNQ1 is required to supply K+ to the luminal binding K+ site of the H+/K+-ATPase, Song et al. (2009) performed an experiment where luminal NaCl was replaced by KCl in 7−day-old KCNQ1−/− and WT mice. This manoeuvre initiated spontaneous acid secretion in KCNQ1−/− mice and fully restored forskolin-stimulated acid secretion to values observed in WT mice. In contrast, acid secretion could not be rescued in adult KCNQ1−/− mice, which is consistent with the severely abnormal gastric morphology that is observed in these mice. Not only do adult KCNQ1−/− mice spontaneously secrete HCO3− rather than H+ under basal conditions, but neither forskolin nor KCl could restore acid secretion. These findings argue against other channels/transporters being involved in apical K+ exit, which could be upregulated in KCNQ1−/− mice as a compensatory consequence of the lack of KCNQ1. In contrast to the massive ultrastructural changes reported in adult KCNQ1−/− gastric mucosa, no changes were found in regard to mucosal thickness or parietal cell number in young KCNQ1−/− mice. Further, while there were no differences in H+/K+-ATPase localization and expression between genotypes, H+/K+-ATPase mRNA expression was shown to be downregulated by 50% in KCNQ1−/− compared to WT mucosa. The reason for this finding remains speculative, but may indicate that mRNA expression levels in parietal cells are not predictive for protein expression levels. On the other hand, even at an age of 7 days, parietal cells in KCNQ1−/− mice already show degenerative changes. Further, electron microscopy studies revealed that parietal cells of KNCQ1−/− mice contain amorphous material of varying electron density and/or vesicular structures surrounded by an electron-dense material which could resemble autophagosomes. Taken together, these findings indicate that KCNQ1 has an essential role in the normal development of parietal cells, cell survival and functional morphogenesis of the gastric mucosa. In our eyes this implies a very dominant role for KCNQ1, especially in parietal cell development. It would be interesting to determine the time course or onset of the observed ultrastructural changes in the gastric mucosa. An excellent model would be to create an inducible conditional knockout for KCNQ1 in parietal cells, in which the role of KNCQ1 in K+ recycling and gastric acid secretion in mice with normal stomach anatomy could be studied. Additionally, inducible deletion of KCNQ1 would help clarify the time course of morphological changes that occur in the parietal cell and/or gastric mucosa. Recently it was shown that the K+/2Cl− cotransporter KCC4 is present in the apical membrane of parietal cells under resting conditions. Song et al. (2009) speculated that KCC4 may provide K+ for K+ recycling as well as secretory Cl− during unstimulated acid secretion. Further studies are needed to define the role of KCC4 in these processes. An interesting experiment would be to use the KCC4 inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid) under basal and stimulated conditions in WT and KCNQ1−/− mice to determine if there is a significant portion of K+ recycling mediated by KCC4. Conversely, 293B could be used in a similar experiment in KCC4−/− mice. In conclusion, the importance of KCNQ1 in gastric acid secretion has been clearly demonstrated by the fact that pharmacological inhibition and genetic knockout result in inhibition of acid secretion. However, it was still debatable whether KCNQ1 channels were the only apical K+ efflux pathway. By utilizing isolated gastric mucosa from very young KCNQ1−/− mice, Song et al. (2009) were able to definitively show that KCNQ1 is the essential apical K+ channel that provides K+ to the extracellular K+ binding site of the H+/K+-ATPase during gastric acid secretion, and that no other parietal cell K+ channels or transporters can adequately substitute for this function.