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

Key points: Receptor-operated activation of TRPC4 cation channels requires G_i/o proteins and phospholipase-Cδ1 (PLCδ1) activation by intracellular Ca²⁺ . Concurrent stimulation of the G_q/11 pathway accelerates G_i/o activation of TRPC4, which is not mimicked by increasing cytosolic Ca²⁺ . The kinetic effect of G_q/11 was diminished by alkaline intracellular pH (pH_i ) and increased pH_i buffer capacity. Acidic pH_i (6.75-6.25) together with the cytosolic Ca²⁺ rise accelerated G_i/o -mediated TRPC4 activation. Protons exert their facilitation effect through Ca²⁺ -dependent activation of PLCδ1. The data suggest that the G_q/11 -PLCβ pathway facilitates G_i/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 G_q/11 and G_i/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 G_i/o proteins, with a co-dependence on phospholipase-Cδ1 (PLCδ1). The G_i/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 G_i/o -coupled μ opioid receptor, we investigated the role of intracellular protons on G_i/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 G_i/o and G_q/11 co-stimulation induces a faster activation of TRPC4 than G_i/o activation alone and highlights again the critical role of PLCδ1 in TRPC4 gating.