Mechanism of Regulation of Gi/o-Mediated TRPC4 Activation by Intracellular Protons

Abstract

Transient Receptor Potential Canonical 4 (TRPC4) protein forms non-selective cation channels activated downstream from receptors that signal through heterotrimeric G proteins. Although the receptor-operated TRPC channel activation has mostly been attributed to Gq/11 proteins or receptor tyrosine kinases through stimulation of phospholipase C (PLC) isoforms β and γ, or internal Ca2+ store depletion, our recently work suggests that TRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o proteins, with a codependence on PLCδ1. We showed that the Gi/o-mediated TRPC4 activation is dually dependent on and bimodally regulated by phosphatidylinositol 4, 5-bisphosphate (PIP2), the substrate hydrolyzed by PLC, and intracellular Ca2+, the level of which is increased following PLC stimulation due to inositol 1,4,5-trisphosphate induced internal Ca2+ release and Ca2+ influx through TRPC4 itself. As a byproduct of PLC-mediated PIP2 hydrolysis, protons have been shown to play an important role in the activation of Drosophila TRP channels. However, how intracellular pH affects mammalian TRPC channels was unclear. Using patch-clamp recordings of HEK293 cells heterologously coexpressing mouse TRPC4β and Gi/o-coupled µ opioid receptor, here we investigated the effect of intracellular protons on Gi/o-mediated TRPC4 activation. We found that lowering cytosolic pH greatly accelerated the rate of TRPC4 activation without altering the maximal current density and this effect was dependent on intracellular Ca2+ elevation. However, protons did not accelerate channel activation via a direct action on TRPC4, as shown by the inhibitory effect of low pH on channel activation by Englerin A, a direct TRPC4 agonist. We propose that protons exert their effect through sensitization of PLCδ1 to Ca2+, which in turn promotes TRPC4 opening and further PLCδ activities via a positive feedback mechanism.