Regulation von TRPM3 durch α2-Adrenorezeptoren in pankreatischen β-Zellen

TRPM3 ist ein Ca2+-permeabler, nichtselektiver Kationenkanal der TRP-Familie, der durch das endogene Neurosteroid Pregnenolonsulfat aktiviert werden kann. Eine funktionelle Expression des Kanals konnte unter anderem in pankreatischen β-Zellen nachgewiesen werden. Eine Aktivierung von TRPM3 in β-Zell...

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Bibliographic Details
Main Author: Mohr, Florian
Contributors: Oberwinkler, Johannes (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
Online Access:PDF Full Text
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TRPM3 is a Ca2+ permeable nonselective cation channel of the transient receptor potential melastatin channel family. The channel is expressed in pancreatic β cells and can be activated by the endogenous neurosteroid pregnenolone sulphate. The activation of TRPM3 in pancreatic β cells leads to calcium influx followed by an enhanced glucose-stimulated insulin release. However, the physiological functions of TRPM3 in pancreatic β cells are not well understood so far. Insulin release in β cells is subject to strong and diverse regulation and can be adapted to different physiological conditions. Many of these regulations are mediated by G-protein coupled receptors (GPCR). It is well known that various TRP channels and most of the ion channels in β cells are regulated by GPCR. The question arose whether TRPM3 activity in β cells is also subject to such a regulation or modulation. Indeed, in primary pancreatic β cells and rat insulinoma cells (Ins1) noradrenaline strongly and reversibly inhibits the TRPM3 activity and pharmacological experiments revealed that the inhibition is mediated via α2-adrenoreceptors. Intrigued by this effect we decided to take a closer look at the intracellular signaling cascades that mediates the TRPM3 inhibition in pancreatic β cells. Experiments with pertussis toxin (PTX), an inhibitor of Gi/o-proteins, revealed that the mechanism is Gi/o-protein coupled. However, we could rule out a classical regulation by alteration of intracellular cAMP levels. Also a direct interaction of Gαi/o subunits with the channel could be excluded through overexpression experiments. By contrast, overexpression of βγ-subunits leads to a strong inhibition of the TRPM3 activity. But only β1 or β2 co-expressed with a γ-subunit were able to strongly inhibit the channel. Furthermore Ins1 cells treated with mSIRK (a membrane permeable peptide that induces a release of βγ-subunits) showed a strong reduction of the TRPM3 activity. Additional experiments with βγ-scavenging peptides (myr-phosducin or myr-βARKct) also led to a reduction of the inhibitory noradrenaline effect. A participation of PLC, PKC, PLA2 and soluble cytosolic factors in the signaling pathway that induces the inhibition of TRPM3 could be largely excluded in further experiments, indicating that βγ-subunits could perhaps directly induce the inhibition of the channel. It turned out that not all TRPM3 splice variants show a α2-adrenoreceptor mediated inhibition or decrease of activity after overexpression of βγ-subunits. It seems that a sequence of 10 amino acids (coded by exon 17) is required for the channel inhibition, since splice variants without this sequence (TRPM3α4 and TRPM3α5) were insensitive to α2-adrenoreceptor mediated inhibition and overexpression of βγ-subunits and showed no reduction of the channel activity. This study clearly shows that TRPM3 is strongly regulated by α2-adrenoreceptors in β cells. Indicating that TRPM3 may play an important part in pancreatic β cells which necessitates strict sympathetic regulation. The results obtained in this study contribute to a better understanding of the function of TRPM3 in β cells. Furthermore, the discovery that TRPM3 can be modulated via Gαi/o-coupled GPCR provides a basis for further investigation of the role, function and regulation of TRPM3 in other tissues.