Channels and Transporters in Zymogen Granule Membranes and their Role in Granule Function: Recent Progress and a Critical Assessment Frank Thévenod Institute of Physiology, Pathophysiology & Toxicology, Centre for Biomedical Training and Research (ZBAF), Faculty of Health, University of Witten/Herdecke, D-58453 Witten, Germany e-mail: [email protected] Version 1.0, January 6, 2015 [DOI: 10.3998/panc.2015.1] - Abstract ClC Cl channels and the vesicular nucleotide transporter SLC17A9 in ZG is less strong. To Secretory granules are located at the apex of better understand the function of these proteins in pancreatic acinar cells. Secretagogues bind to the secretory pathway further studies are needed. their receptors at the basolateral membrane of acinar cells and trigger the activation of 1. Introductory Remarks intracellular signaling pathways to elicit fusion of secretory granules with the apical plasma A review on the topic of pancreatic zymogen membrane that is followed by exocytosis of granule (ZG) channels and transporters and their digestive pro-enzymes (the “zymogens”) into the function is timely as the last exhaustive review lumen. This regulated discharge of stored appeared in 2002 (107) and is manageable macromolecules is accompanied by the secretion because of the limited number of publications that of solutes and water to the cell exterior to hydrate had been published in the 12 years since then. these protein-rich secretory products. Previous These circumstances have allowed me to carry functional and pharmacological studies in out an in-depth and critical analysis of the pancreatic acinar cells and zymogen granules published data. The advent of the post-genomic (ZG) had suggested that ion channels and era had raised hopes that – similar to other areas transporters are expressed in the membrane of of cell biology – a large number of ZG transport ZG where they contribute to maturation, fusion, proteins would be identified and their role in exocytosis and/or fluidization of zymogens. This pancreatic secretion would be elucidated. Indeed, chapter reviews studies that have been largely recent studies have combined functional and published in the postgenomic era and combined molecular approaches to characterize ZG biochemical, immunological, electrophysiological, channels and transporters and their role in pharmacological, and/or occasionally knockout pancreas physiology. Yet, the fact that only a very methodologies to identify cloned transporters and limited number of studies have been published in ion channels in the membrane of ZG. Available this area of research is surprising as there have experimental evidence indicates the presence of been tremendous developments of knowledge several ion channel and transporter proteins in ZG and methodologies available to investigate the membranes (aquaporins, vacuolar-type H+- molecular and cellular biology and physiology of ATPase, zinc influx transporter SLC30A2). The the pancreas (122). + evidence for the K channels Kv7.1 and Kir6.1, for This work is subject to an International Creative Commons Attribution 4.0 license. Figure 1. The model summarizes current evidence (strong or weak) for the presence of ion channels and transporters in zymogen granules (ZG) of pancreatic acinar cells. The Figure also describes relevant ion concentrations in mature ZG and emphasizes the decreasing lumen acidity during maturation along the compartments of the secretory pathway that participate in ZG maturation (for further details, see text). Moreover, a better understanding of the the role played by ZG in this process seems to be physiology of pancreatic acinar cell secretion and a prerequisite to comprehend the pathogenesis of pancreatic disorders, such as pancreatitis, cystic point) in suspensions of isolated ZG (107). Such fibrosis or cancer (65). work has provided important 'background' functional information for interpreting the post- In the following paragraphs, I first review genomic work (see below). significant advances in the characterization of ZG channels and transporters in the last decade and A key advance in the last decade has been the discuss their putative role in pancreatic acinar widespread availability of proteomics that identify secretion (Figure 1). The review ends on a a large number of expressed proteins personal assessment of possible causes for the simultaneously. The use of proteomics for current problems and apparent stagnation of this analysis of ZG and their membranes has field of research and makes suggestions how to propelled the identification of channels and overcome these difficulties. transporters of pancreatic ZG although various caveats need to be contemplated. Several 2. Proteomics proteomic studies have been published that used highly purified ZG membranes (ZGM) to identify In the past, ion pathways in ZG were either cloned transporters and channel proteins (9, 18, characterized in permeabilized acini in which the 19, 96). In most of these studies, granules were effect of the ionic composition of the “extended disrupted by different techniques and membranes cytosol” on stimulated secretion was investigated were subjected to carbonate and/or bromide and/or by recording macroscopic ion fluxes using extraction, which is a standardized and reliable an osmotic swelling assay (and lysis as an end- procedure to obtain pure membranes without 2 peripheral proteins (14). A critical step in this lysosomes where it operates as a nonselective isolation procedure represents the initial cation channel that is inhibited by luminal acidity “purification” of ZG by either differential or while increasing the luminal pH in the presence of continuous Percoll gradient centrifugation. These ATP causes cation channel activation (49). These methods yield fractions that are enriched 4-8- functional properties in a P2X4 ATP receptor that times in α-amylase compared to homogenate, would be located in ZG (if it is in fact present there which is close to the theoretical limit of purity (77). because lysosomes easily contaminate ZGM) are Yet, it is practically impossible to avoid attractive for ZG exocytosis considering the fact contaminations by lysosomes (96), plasma that maturing ZG lose their acidity (see the membranes (110) or membranes from other paragraph on Vacuolar-type H+-ATPase) and that (disrupted) organelles (see electron micrographs the P2X4 cation channel is activated by ATP in references (14, 110, 128)). These contaminants concentrations that have been measured in ZG are, however, a major draw-back for proteomic (see paragraph on the Vesicular nucleotide analyses (as well as for electrophysiological transporter SLC17A9). Nonetheless, the detection studies; see the paragraph on K+ Channels) of the P2X4 ATP receptor offers an example of because even minimal contaminations by the potentially novel information on ZG physiology membranes originating from other organelles or that may be derived from proteomic approaches. plasma membranes may be prominent in a Hence, P2X4 is a promising candidate for future proteomic analysis of ZGM because of proteins studies on ZG function. that are highly expressed in these contaminating membranes. This issue needs to be considered in 3. Channels particular for “mitochondrial” proteins such as voltage-dependent anion channels (VDACs) and H2O Channels subunits of ATP-synthase that have been It is a long-standing observation that isolated ZG detected in ZGM in two independent studies (9, and other secretory granules suspended in 19), or “plasma membrane” proteins, such as the isotonic KCl buffers remain stable for hours (7, α-subunit of Na+/K+-ATPase (96). Hence, these 26). Considering the packaging and condensation observations need to be taken with caution. In of a variety of osmotically active ions, small addition, detection of a particular channel or organic molecules and macromolecules in the transporter in ZGM may not infer any lumen of ZG during maturation and bearing in physiological/functional significance of that mind that granular Cl- and K+ concentrations are identified protein in mature ZG (see the paragraph lower than in the cytosol (83, 97), ZGs must + + on Vacuolar-type H -ATPase). Finally, it should exhibit low basal permeabilities for H2O, K and be born in mind that several cloned channel Cl- in situ and in vitro. But it has long been known proteins and transporters that have been that addition of cation or anion ionophores to identified by functional or immunological isolated ZG suspended in iso-osmotic KCl buffers approaches have not been detected by proteomic elicits granular swelling (26). The ionophore is analyses. This may be due to a relative thought to generate an electrochemical insensitivity of proteomic approaches because of membrane potential that activates endogenous a very low expression level of these proteins in conductive pathways for the counterions and ZGM. allows entry of osmotically active KCl into the granular lumen (26, 108). H2O follows and Interestingly, among the putative novel induces swelling (and lysis) implying that H2O transporters and channels identified in ZGM by permeable pathways, e.g. aquaporins, are Rindler and coworkers (96) the ligand gated ATP present in ZGM. receptor P2X4 deserves special attention because P2X4 has also been recently identified in 3 The expression and function of aquaporins in membranes of ZGs near the rER. Furthermore, pancreas has been recently reviewed (29). AQP12 knockout mice were more prone to Aquaporin 1 (AQP1) has been identified in rat caerulein-induced