HCO3− Transport through Anoctamin/Transmembrane Protein ANO1/TMEM16A in Pancreatic Acinar Cells Regulates Luminal pH

Yanfeng Han,Annette M Shewan,Peter Thorn

doi:10.1074/jbc.m116.750224

Yanfeng Han, Annette M Shewan + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m116.750224

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Abstract

The identification of ANO1/TMEM16A as the likely calcium-dependent chloride channel of exocrine glands has led to a more detailed understanding of its biophysical properties. This includes a calcium-dependent change in channel selectivity and evidence that HCO3 (-) permeability can be significant. Here we use freshly isolated pancreatic acini that preserve the luminal structure to measure intraluminal pH and test the idea that ANO1/TMEM16A contributes to luminal pH balance. Our data show that, under physiologically relevant stimulation with 10 pm cholesystokinin, the luminal acid load that results from the exocytic fusion of zymogen granules is significantly blunted by HCO3 (-) buffer in comparison with HEPES, and that this is blocked by the specific TMEM16A inhibitor T16inh-A01. Furthermore, in a model of acute pancreatitis, we observed substantive luminal acidification and provide evidence that ANO1/TMEM16A acts to attenuate this pH shift. We conclude that ANO1/TMEM16A is a significant pathway in pancreatic acinar cells for HCO3 (-) secretion into the lumen.

Highlights

Luminal pH dysregulation in exocrine organs is a key factor in diseases like cystic fibrosis and acute pancreatitis [1, 2]
Our results show that the likely major action of HCO3Ϫ buffer on luminal pH is via TMEM16A, leading us to conclude that TMEM16A is a transport pathway for apical HCO3Ϫ exit that can significantly alter luminal pH under physiologically relevant stimulation
The major finding of our study is that, under physiologically relevant stimulation, HCO3Ϫ exit through the apical ANO1/TMEM16A channel of pancreatic acinar cells is a significant regulator of intraluminal pH. This observation builds on the biophysical characterization of ANO1/ TMEM16A and provides evidence that acinar cells play an active role in regulating the pH of the pancreatic lumen

Summary

Introduction

Luminal pH dysregulation in exocrine organs is a key factor in diseases like cystic fibrosis and acute pancreatitis [1, 2]. TMEM16A as the likely candidate for the apical calcium-dependent chloride channel in exocrine acinar cells [7,8,9,10,11] and some recent work on its control [12] highlight the possibility that it may act as an apical HCO3Ϫ exit pathway and could be an additional component in pancreatic luminal pH regulation [13]. We used 10 pM cholesystokinin (CCK) [22] to study the physiological response and 10 nM CCK to model the cell responses in acute pancreatitis In both cases we observed that the stimulated exocytosis of zymogen granules release their acid content into the lumen. At both normal and high levels of stimulation, ANO1/TMEM16A provides an HCO3Ϫ exit pathway in acinar cells that regulates luminal pH. B, plots of fluorescence over time (baseline subtracted, n Ն 33 events, n ϭ 3ϩ animals) within regions of interest (A, arrows) over the region of the fusing granules showing that both

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