Phosphatidylinositol 4,5-bisphosphate and loss of PLCγ activity inhibit TRPM channels required for oscillatory Ca2+signaling

Abstract

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+oscillations that control muscle contractions required for defecation. Two highly Ca2+-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLCγ in a common signaling pathway that regulates IP3-dependent intracellular Ca2+release. A second PLC, PLCβ, is also required for IP3-dependent Ca2+oscillations, but functions in an independent signaling mechanism. PLCγ generates IP3that regulates IP3receptor activity. We demonstrate here that PLCγ via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) also regulates GON-2/GTL-1 function. Knockdown of PLCγ but not PLCβ activity by RNA interference (RNAi) inhibits channel activity ∼80%. Inhibition is fully reversed by agents that deplete PIP2levels. PIP2added to the patch pipette has no effect on channel activity in PLCγ RNAi cells. However, in control cells, 10 μM PIP2inhibits whole cell current ∼80%. Channel inhibition by phospholipids is selective for PIP2with an IC50value of 2.6 μM. Elevated PIP2levels have no effect on channel voltage and Ca2+sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP2by PLCγ functions in the activation of both the IP3receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca2+influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP3receptor function. PIP2-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca2+influx with PLCγ and IP3receptor activity as well as intracellular Ca2+store depletion.