title: Phosphatidylinositol 4,5-bisphosphate and loss of PLCgamma activity inhibit TRPM channels required for oscillatory Ca2+ signaling.
abstract: The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) oscillations that control muscle contractions required for defecation. Two highly Ca(2+)-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLCgamma in a common signaling pathway that regulates IP(3)-dependent intracellular Ca(2+) release. A second PLC, PLCbeta, is also required for IP(3)-dependent Ca(2+) oscillations, but functions in an independent signaling mechanism. PLCgamma generates IP(3) that regulates IP(3) receptor activity. We demonstrate here that PLCgamma via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) also regulates GON-2/GTL-1 function. Knockdown of PLCgamma but not PLCbeta activity by RNA interference (RNAi) inhibits channel activity approximately 80%. Inhibition is fully reversed by agents that deplete PIP(2) levels. PIP(2) added to the patch pipette has no effect on channel activity in PLCgamma RNAi cells. However, in control cells, 10 microM PIP(2) inhibits whole cell current approximately 80%. Channel inhibition by phospholipids is selective for PIP(2) with an IC(50) value of 2.6 microM. Elevated PIP(2) levels have no effect on channel voltage and Ca(2+) sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP(2) by PLCgamma functions in the activation of both the IP(3) receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca(2+) influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP(3) receptor function. PIP(2)-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca(2+) influx with PLCgamma and IP(3) receptor activity as well as intracellular Ca(2+) store depletion.
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Xing
authors.forename: Kevin
authors.initials: K
authors.lastname: Strange
mesh_terms: D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D025461:Feedback, Physiological; D050980:GATA Transcription Factors / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D007413:Intestinal Mucosa / Q000201:enzymology*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D010507:Periodicity; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D054799:Phospholipase C beta / Q000235:genetics / Q000378:metabolism; D051966:Phospholipase C gamma / Q000172:deficiency* / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; C096869:ELT-1 protein, C elegans; D050980:GATA Transcription Factors; C487576:GON-2 protein, C elegans; C504382:GTL-1 protein, C elegans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels; D019269:Phosphatidylinositol 4,5-Diphosphate; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; C517632:Egl-8 protein, C elegans; D054799:Phospholipase C beta; D051966:Phospholipase C gamma