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Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

ABSTRACT: The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.

SUBMITTER: Thevenod F

PROVIDER: S-EPMC1149665 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Project description:The zymogen granule is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and is a classic model for studying secretory granule function. Our long term goal is to develop a comprehensive architectural model for zymogen granule membrane (ZGM) proteins that would direct new hypotheses for subsequent functional studies. Our initial proteomics analysis focused on identification of proteins from purified ZGM (Chen, X., Walker, A. K., Strahler, J. R., Simon, E. S., Tomanicek-Volk, S. L., Nelson, B. B., Hurley, M. C., Ernst, S. A., Williams, J. A., and Andrews, P. C. (2006) Organellar proteomics: analysis of pancreatic zymogen granule membranes. Mol. Cell. Proteomics 5, 306-312). In the current study, a new global topology analysis of ZGM proteins is described that applies isotope enrichment methods to a protease protection protocol. Our results showed that tryptic peptides of ZGM proteins were separated into two distinct clusters according to their isobaric tag for relative and absolute quantification (iTRAQ) ratios for proteinase K-treated versus control zymogen granules. The low iTRAQ ratio cluster included cytoplasm-orientated membrane and membrane-associated proteins including myosin V, vesicle-associated membrane proteins, syntaxins, and all the Rab proteins. The second cluster having unchanged ratios included predominantly luminal proteins. Because quantification is at the peptide level, this technique is also capable of mapping both cytoplasm- and lumen-orientated domains from the same transmembrane protein. To more accurately assign the topology, we developed a statistical mixture model to provide probabilities for identified peptides to be cytoplasmic or luminal based on their iTRAQ ratios. By implementing this approach to global topology analysis of ZGM proteins, we report here an experimentally constrained, comprehensive topology model of identified zymogen granule membrane proteins. This model contributes to a firm foundation for developing a higher order architecture model of the ZGM and for future functional studies of individual ZGM proteins.

Project description:Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 ?M and 55 ± 2 ?M, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 ?M and 20 ± 1 ?M, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 ?M. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.

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Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

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