TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells

Abstract

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca2+-activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca2+ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca2+-activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca2+-dependent depolarization of PACs from a resting membrane potential of −44.4 ± 2.9 to −27.7 ± 3 mV. Furthermore, we showed that Ca2+ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca2+ transients partially rely on Ca2+ influx in PACs, the role of TRPM4 was also assessed on Ca2+ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca2+ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca2+. In conclusion, TRPM4 links intracellular Ca2+ signaling to membrane potential as a negative feedback regulator of Ca2+ entry in PACs. Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca2+-activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca2+ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca2+-activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca2+-dependent depolarization of PACs from a resting membrane potential of −44.4 ± 2.9 to −27.7 ± 3 mV. Furthermore, we showed that Ca2+ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca2+ transients partially rely on Ca2+ influx in PACs, the role of TRPM4 was also assessed on Ca2+ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca2+ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca2+. In conclusion, TRPM4 links intracellular Ca2+ signaling to membrane potential as a negative feedback regulator of Ca2+ entry in PACs. Pancreatic acinar cells (PACs) are the major cell types of the exocrine pancreas. They are responsible for secretion of digestive enzymes and primary fluid. Stimulation by endogenous secretagogues, such as acetylcholine and cholecystokinin, causes inositol 1,4,5-trisphosphate (IP3) generation, and consequent Ca2+ release from the endoplasmic reticulum (ER) through the IP3 receptor (IP3R) Ca2+ channels. The subsequent increase in intracellular Ca2+ concentration ([Ca2+]i) triggers the exocytosis of digestive enzymes (1Streb H. Irvine R.F. Berridge M.J. Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.Nature. 1983; 306: 67-69Crossref PubMed Scopus (1762) Google Scholar, 2Streb H. Heslop J.P. Irvine R.F. Schulz I. Berridge M.J. Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells.J. Biol. Chem. 1985; 260: 7309-7315Abstract Full Text PDF PubMed Google Scholar, 3Ito K. Miyashita Y. Kasai H. 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PubMed Scopus (24) Google the current and that is an for TRPM4 the of Ca2+ both cation and currents in not the current in pancreatic acinar cells. current of currents of a cell control and the application of between and with Ca2+ in the data are in 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic image current was also in PACs from in which the the TRPM4 was R. J. M. I. P. M. cell activation and in the nonselective cation channel 2007; PubMed Scopus Google using the Although, the current was higher to to increase and was not sensitive to These results suggest that TRPM4 is in PACs in significant In to the of TRPM4 current on membrane using the current The membrane potential was −44.4 ± 2.9 control and the membrane depolarized to −27.7 ± 3 was The membrane potential to the resting ± the was with and on we that membrane in a Ca2+-dependent the activation of Furthermore, we that the driving force for Ca2+ influx depolarized membrane is enough to significantly Ca2+ this Ca2+ was in of PACs, which exposed to with of which is to a physiological with cholecystokinin R. E. Sutton R. Petersen O.H. and exhibit on calcium signaling, zymogen and cell in pancreatic acinar cells.Am. J. Physiol. Gastrointest. Liver Physiol. 2009; PubMed Scopus Google Scholar). in and the of Ca2+ using control and TRPM4 KO cells as application of evoked of [Ca2+]i in and Ca2+ spikes between and of Ca2+ entry was to to the Ca2+ signaling by this D.I. Lawrie A.M. Gallacher D.V. and cholecystokinin induce patterns of calcium signals in pancreatic acinar 1991; 12: PubMed Scopus (95) Google Scholar). the amplitude of Ca2+ spikes in control PACs was in and the was higher in in the of TRPM4 KO Ca2+: ± Ca2+: ± TRPM4 KO Ca2+: ± and TRPM4 KO Ca2+: ± These data that Ca2+ entry is significant in TRPM4 KO PACs but not in control cells. the in control and TRPM4 KO PACs the in that the Ca2+ of was the of in both types of cells. the is to to the of intracellular followed a but the not significantly not the temporal of Ca2+ transients not in control and TRPM4 KO PACs. In conclusion, the between control and KO Ca2+ spikes in that TRPM4 is in the negative feedback regulation of Ca2+ entry in PACs. the role of TRPM4 using in a was not of a that Ca2+ oscillations in not that Ca2+ release in PACs. The that TRPM4 is in the negative feedback regulation of Ca2+ entry was in to cause significant Ca2+ from the in to PACs stimulated with for in sustained Ca2+ signals with of is an of and Ca2+ of the of the was to a containing Ca2+, which in a of [Ca2+]i, to the activation of The amplitude of the was with in TRPM4 KO PACs and found to significantly higher in KO cells ± TRPM4 ± was by using in to cause in a that with the secretagogue of was in to induce Ca2+ from the In the beginning of [Ca2+]i which was followed by a that the and Ca2+ was from the intracellular by The amplitude of Ca2+ signals was not significantly between and KO PACs ± ± and ± the was by which in a increase in [Ca2+]i in the of or using TRPM4 KO PACs. In was to for the of TRPM4 the was and the was not Ca2+ release or the of the of of that the and amplitude of the of was higher in TRPM4 KO PACs with In significantly the of not the of the and ± ± ± ± ± ± These data are in with in and that TRPM4 is a negative feedback regulator of Ca2+ entry in PACs. In this study, we the that the TRPM4 current PACs in a Ca2+-dependent and as a negative feedback regulator of Ca2+ entry physiological However, data may also pathological Ca2+ overload of PACs is to the pathological leading to intracellular zymogen activation, self-digestion, and acute pancreatitis (16Ward J.B. Petersen O.H. Jenkins S.A. Sutton R. Is an elevated concentration of acinar cytosolic free ionised calcium the trigger for acute pancreatitis?.Lancet. 1995; 346: 1016-1019Crossref PubMed Scopus (152) Google Scholar, 17Gerasimenko J.V. Lur G. Sherwood M.W. Ebisui E. Tepikin A.V. Mikoshiba K. Gerasimenko O.V. Petersen O.H. Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 10758-10763Crossref PubMed Scopus (83) Google Scholar, 18Gerasimenko J.V. Gerasimenko O.V. Petersen O.H. The role of Ca2+ in the pathophysiology of pancreatitis.J. Physiol. 2014; 592: 269-280Crossref PubMed Scopus (93) Google Scholar). As is to sustained and pathological of [Ca2+]i and inhibitors reported to the of acute data the that TRPM4 a role in the pathophysiology of Ca2+ signaling J.V. E. S. S. M. Gerasimenko O.V. Petersen O.H. Ca2+ Ca2+ channel as a potential in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). using of the The and potential of TRPM4 also physiological functions of TRPM4 been in and cells TRPM4 Ca2+ oscillations to sustained elevations of [Ca2+]i and to production in which is in with the that TRPM4 Ca2+ entry by the membrane and the driving force for Ca2+ influx R. J. M. I. P. 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The was with and in The was in of this in a for with The was with The was by by 4 to of through a through a on the of containing and by The was in and by cell in and in Ca2+ In to single acinar cells for the acinar cell to an in Ca2+ and containing for cells with using a cell with Ca2+ sensitive for cells on and to to the with containing and and with a which but of was using a with a was and using an and the was through a and using a The was by A.D. H. of control using Biol. 2014; PubMed Google Scholar, A.D. K. R. control of using Biol. PubMed Scopus Google through an using of with of elicited by and in single cells. of for and as ± currents using an and a a and using a of to from In TRPM4 current with a containing 3 The 4 In current the The extracellular of the from TRPM4 current was using a from to whereas current was between and mV. of the was in potential was using the The cells in whereas the of the was with a containing The was with the with was on a using the A. J. A. A. acid of via acid receptor PubMed Scopus Google Scholar). was using was to the and of was using from was using the for for for for inositol 1,4,5-trisphosphate receptor type for type for type for for and for and the As internal of the The amount of the was to the using the was from was from and from was in or in are as the of cells from and are as ± was by using or with using and with significant was than The of the of of cells in the of Ca2+ and the of cells in as in the to the data are the and The that of with the of this The are to and for The was by the of the for and in the of the of the of was by the which is by the and the The by the and and I. and J. A. G. E. E. E. P. P. R. I. and J. A. G. and J. A. G. E. E. and J. A. R. I. and J. A. J. A. G. E. E. E. P. P. R. and I. and G. and J. A. I. and J. A. P. P. R. I. and J. A. J. A. is a of the of the of of I. was by the of the for and and the of the of R. is by from the and

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