Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels

Background/Aims: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release. Methods: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests. Results: TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose. Conclusion: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release

Analysis of TRPM3 cation channels using knock down and knock out methods

Einen wesentlichen Anteil an der Regulation der intrazellulären Ca2+-Konzentration haben Kationenkanäle aus der Familie der TRP-Proteine. Vermutlich 4 TRP-Proteine bilden homo- und heterooligomere Kanalkomplexe. TRPM3 ist das zuletzt identifizierte Mitglied der TRPM-Subfamilie mit größter Homologie zu TRPM1. Das TRPM3-Gen codiert eine Vielzahl von Spleissvarianten, welche Unterschiede hinsichtlich ihrer Permeabilität für divalente Kationen und ihrer Aktivierbarkeit durch das Steroid Pregnenolonsulfat aufweisen. Ich habe gezeigt, dass TRPM3 vor allem im Plexus choroideus, in ß-Zellen und in der Hypophyse exprimiert wird. Aus Ins-1- und GH3-Zellen sowie aus dem Hirn der Ratte habe ich neben bekannten TRPM3-Varianten 9 bislang unbekannte kloniert. FURA-2 Messungen zeigten, dass Pregnenolonsulfat in Ins-1- und GH3-Zellen einen Ca2+-Einstrom auslöst. Eine Hemmung von TRPM3 mit Hilfe von RNA-Interferenz ebenso wie eine Überexpression von TRPM1 und TRPM31 führte in diesen Zellen zu einer deutlichen Abnahme des Steroid-induzierten Ca2+-Einstroms, was stark darauf hindeutet, dass TRPM3-Kanäle für den Ca2+-Einstrom verantwortlich sind. Um die biologische Funktion von TRPM3 zu untersuchen, habe ich eine TRPM3-defiziente Mauslinie hergestellt. Dabei wurde Exon 24 deletiert, da es die ionenleitende Pore des Kanals codiert. Die angewandte Strategie erlaubt zudem eine zeit- oder gewebespezifische Inaktivierung des TRPM3-Gens sowie die Identifizierung TRPM3-exprimierender Zellen anhand ihrer grünen Fluoreszenz.Cation channels of the TRP family play a substantial role in the regulation of the cytosolic Ca2+ homeostasis. Presumably, TRP channel subunits assemble into homo- or heterotetrameric channel complexes. TRPM3 is the most recent identified member of the TRPM subfamily and shows highest homology to TRPM1. The TRPM3 gene encodes a variaty of splice variants. These variants differ in their permeability for divalent cations and their activation by the steroid pregnenolonesulfate. In this work I showed that TRPM3 is strongly expressed in the choroid plexus, in pancreatic ß-cells and in the pituitary gland. I cloned several variants of TRPM3 from Ins-1 cells, GH3 cells and rat brain. Nine new splice variants were identified. Pregnenolonesulfate induces Ca2+ entry in GH3 and Ins-1 cells as measured with the calciumindicatordye FURA-2. Inhibition of TRPM3 using a RNA interference approach and overexpression of TRPM1 and TRPM31 led to a significant reduction of the steroid induced Ca2+ influx in these cells. These data strongly suggest that TRPM3 channels are responsible for the steroid induced Ca2+ entry in endocrine cells. To analyze the biological functions of TRPM3 channels, I generated a TRPM3 deficient mouse line. Thereby exon 24 was deleted since it encodes the ion conducting pore of the channel. Furthermore, the applied strategy allows a time- or tissue-specific inactivation of the TRPM3 gene and in addition the identification of TRPM3 expressing cells by their green fluorescenc

A background Ca 2+ entry pathway mediated by TRPC1/TRPC4 is critical for development of pathological cardiac remodelling

Aims: Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca2+ signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca2+ homeostasis in cardiomyocytes during fast cytosolic Ca2+ cycling and neurohumoral stimulation leading to hypertrophy is unknown. Methods and results: In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn2+-quench microfluorimetry, we identified a background Ca2+ entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca2+ concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca2+-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure. Conclusions: The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca2+ cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis.Fil: Camacho Londoño, Juan E.. Pharmakologisches Institut; Alemania. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania. German Centre for Cardiovascular Research; AlemaniaFil: Tian, Qinghai. Institut fur Molekulare Zellbiologie; AlemaniaFil: Hammer, Karin. Institut fur Molekulare Zellbiologie; AlemaniaFil: Schröder, Laura. Institut fur Molekulare Zellbiologie; AlemaniaFil: Camacho Londoño, Julia. Experimentelle und Klinische Pharmakologie und Toxikologie; AlemaniaFil: Reil, Jan C.. Universitat des Saarlandes; AlemaniaFil: He, Tao. German Centre for Cardiovascular Research; Alemania. Research Unit Cardiac Epigenetics; Alemania. Tongji Hospital; ChinaFil: Oberhofer, Martin. Institut fur Molekulare Zellbiologie; AlemaniaFil: Mannebach, Stefanie. Experimentelle und Klinische Pharmakologie und Toxikologie; AlemaniaFil: Mathar, Ilka. Pharmakologisches Institut; Alemania. Experimentelle und Klinische Pharmakologie und Toxikologie; AlemaniaFil: Philipp, Stephan E.. Experimentelle und Klinische Pharmakologie und Toxikologie; AlemaniaFil: Tabellion, Wiebke. Institut fur Molekulare Zellbiologie; AlemaniaFil: Schweda, Frank. Universitat Regensburg; AlemaniaFil: Dietrich, Alexander. Walther-Straub-Institut fur Pharmakologie und Toxikologie; AlemaniaFil: Kaestner, Lars. Institut fur Molekulare Zellbiologie; AlemaniaFil: Laufs, Ulrich. Universitat des Saarlandes; AlemaniaFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Transmembrane Signaling Group; AlemaniaFil: Flockerzi, Veit. Experimentelle und Klinische Pharmakologie und Toxikologie; AlemaniaFil: Freichel, Marc. Pharmakologisches Institut; Alemania. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania. German Centre for Cardiovascular Research; AlemaniaFil: Lipp, Peter. Institut fur Molekulare Zellbiologie; Alemani

Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells: ACTIVATION OF EGR-1 EXPRESSION INVOLVING TRPM3, VOLTAGE-GATED CALCIUM CHANNELS, ERK, AND TERNARY COMPLEX FACTORS*

The neurosteroid pregnenolone sulfate acts on the nervous system by modifying neurotransmission and receptor functions, thus influencing synaptic strength, neuronal survival, and neurogenesis. Here we show that pregnenolone sulfate induces a signaling cascade in insulinoma cells leading to enhanced expression of the zinc finger transcription factor Egr-1 and Egr-1-responsive target genes. Pharmacological and genetic experiments revealed that influx of Ca2+ ions via transient receptor potential M3 and voltage-gated Ca2+ channels, elevation of the cytosolic Ca2+ level, and activation of ERK are essential for connecting pregnenolone sulfate stimulation with enhanced Egr-1 biosynthesis. Expression of a dominant-negative mutant of Elk-1, a key regulator of gene transcription driven by a serum response element, attenuated Egr-1 expression following stimulation, indicating that Elk-1 or related ternary complex factors connect the transcription of the Egr-1 gene with the pregnenolone sulfate-induced intracellular signaling cascade elicited by the initial influx of Ca2+. The newly synthesized Egr-1 was biologically active and bound under physiological conditions to the regulatory regions of the Pdx-1, Synapsin I, and Chromogranin B genes. Pdx-1 is a major regulator of insulin gene transcription. Accordingly, elevated insulin promoter activity and increased mRNA levels of insulin could be detected in pregnenolone sulfate-stimulated insulinoma cells. Likewise, the biosynthesis of synapsin I, a synaptic vesicle protein that is found at secretory granules in insulinoma cells, was stimulated in pregnenolone sulfate-treated INS-1 cells. Together, these data show that pregnenolone sulfate induces a signaling cascade in insulinoma cells that is very similar to the signaling cascade induced by glucose in β-cells

Transient Receptor Potential Melastatin 1 (TRPM1) Is an Ion-conducting Plasma Membrane Channel Inhibited by Zinc Ions*

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic β cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents