Regulation of the Ion Channel TRPM3 by Phosphoinositides

Abstract

TRPM3 belongs to the Melastatin family of Transient Receptor Potential (TRP) ion channels. It is a Ca2+ permeable outwardly rectifying nonselective cation channel. TRPM3 is expressed in sensory neurons, brain, pancreas, kidney and vascular smooth muscle. In sensory dorsal root ganglion (DRG) neurons it was shown to function as a sensor for noxious heat. TRPM3-/- mice have decreased ability to detect noxious heat. In DRG neurons as well as pancreatic beta cells, the neurosteroid pregnenolone sulfate has been shown to activate TRPM3.Many members of the TRP family are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Thus we decided to test if TRPM3 is also regulated by PtdIns(4,5)P2. PtdIns(4,5)P2 is predominantly present at the cytoplasmic face of the plasma membrane and it is an important signaling molecule. In the current study we modulated the levels of PtdIns(4,5)P2 in Xenopus laevis oocytes and HEK cells heterologously expressing TRPM3. In inside-out patches, TRPM3 currents ran down rapidly after excision. This current could be restored by the exogenous application of water soluble diC8 PtdIns(4,5)P2 and the naturally occurring arachidonyl-stearyl (AASt) PtdIns(4,5)P2. Also, application of MgATP to excised inside-out patches reactivated the TRPM3 current. This effect of MgATP was inhibited by LY294002 at a concentration where it inhibits phosphatidylinositol 4 kinase (300 µM) but not at a concentration where it inhibits phosphatidylinositol 3 kinase (10 µM). This suggests that MgATP acted via replenishing PtdIns(4,5)P2 and PtdIns(4)P. In addition, inducing the activity of PtdIns(4,5)P2-5-phosphatases in whole-cell patch clamp experiments also decreased TRPM3 currents. Overall our data collected through excised inside-out and whole-cell patch clamp measurements suggest that TRPM3 requires PtdIns(4,5)P2 as a cofactor.