Intracellular acidification facilitates receptor-operated TRPC4 activation through PLCδ1 in a Ca2+ -dependent manner.

KEY POINTS: Receptor-operated activation of TRPC4 cation channels requires Gi/o proteins and phospholipase-Cδ1 (PLCδ1) activation by intracellular Ca2+ . Concurrent stimulation of the Gq/11 pathway accelerates Gi/o activation of TRPC4, which is not mimicked by increasing cytosolic Ca2+ . The kinetic effect of Gq/11 was diminished by alkaline intracellular pH (pHi ) and increased pHi buffer capacity. Acidic pHi (6.75-6.25) together with the cytosolic Ca2+ rise accelerated Gi/o -mediated TRPC4 activation. Protons exert their facilitation effect through Ca2+ -dependent activation of PLCδ1. The data suggest that the Gq/11 -PLCβ pathway facilitates Gi/o activation of TRPC4 through hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2 ) to produce the initial proton signal that triggers a self-propagating PLCδ1 activity supported by regenerative H+ and Ca2+ . The findings provide novel mechanistic insights into receptor-operated TRPC4 activation by coincident Gq/11 and Gi/o pathways and shed light on how aberrant activation of TRPC4 may occur under pathological conditions to cause cell damage. ABSTRACT: Transient Receptor Potential Canonical 4 (TRPC4) forms non-selective cation channels activated downstream from receptors that signal through G proteins. Our recent work suggests that TRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o proteins, with a co-dependence on phospholipase-Cδ1 (PLCδ1). The Gi/o -mediated TRPC4 activation is dually dependent on and bimodally regulated by phosphatidylinositol 4,5-bisphosphate (PIP2 ), the substrate hydrolysed by PLC, and intracellular Ca2+ . 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 remains obscure. Here, using patch-clamp recordings of HEK293 cells heterologously co-expressing mouse TRPC4β and the Gi/o -coupled μ opioid receptor, we investigated the role of intracellular protons on Gi/o -mediated TRPC4 activation. We found that acidic 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 by directly acting upon TRPC4. We additionally demonstrated that protons exert their effect through sensitization of PLCδ1 to Ca2+ , which in turn promotes PLCδ1 activity and further potentiates TRPC4 via a positive feedback mechanism. The mechanism elucidated here helps explain how Gi/o and Gq/11 co-stimulation induces a faster activation of TRPC4 than Gi/o activation alone and highlights again the critical role of PLCδ1 in TRPC4 gating.