RASD1 Independently Activates TRPC4 through Gαi of GPCR

Abstract

Canonical transient receptor potential (TRPC) channels have six transmembrane (6-TM) domains and are Ca2+-permeable and non-selective cation channels. It is generally speculated that TRPC channels are activated by stimulation of Gq-PLC-coupled receptors and oxidation. Activator of G-protein signaling1 (AGS1 or RASD1), the ras-related protein, interacts with Gi/Go and activates heterotrimeric G-protein signaling systems independent of G-protein-coupled receptor (GPCR). It is previously reported that AGS1 is related to GIRK channel and Ca2+ channel. However it is unknown whether AGS1 is associated with TRPC channels. We assumed that AGS1 might regulate TRPC4 channel, since AGS1 interacts with Gi/Go and TRPC4 is activated by Gi/o subunits. Here, we measured whole cell current of TRPC4/5 after the co-expression of TRPC4 or TRPC5 with constitutively active form of small GTPases in HEK293 cells. AGS1 (CA) mutant (Q to L) activated TRPC4 (38.8 ± 7.2 pA/pF) without GTPγS and independently of GPCR. Pertussis toxin (PTX), Gαi specific inhibitor, blocked RASD1-activated TRPC4 current (3.4±1.6 pA/pF). When co-expressed with dominant negative Gαi protein subtype, TRPC4 activation by RASD1 was completely inhibited. With previous report that TRPC4 are activated primarily by selective Gαi subunits rather than Gαq, these results suggest that AGS1 activates TRPC4 channel through modulating Gαi subunits and AGS1 is a new activator for TRPC4 channel.