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

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-C delta 1 (PLC delta 1). 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 TRPC4 beta and the G(i/o)-coupled mu 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 PLC delta 1 to Ca2+, which in turn promotes PLC delta 1 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 PLC delta 1 in TRPC4 gating.