Close spatio-association of the transient receptor potential canonical 4 (TRPC4) channel with G alpha in TRPC4 activation process

摘要

TPRC channels are Ca2 ^-permeable, nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GP-CRs). TRPC4 is commonly assumed to be activated by Gq/phospho-lipase C-coupled receptors. However, the other molecular mechanisms by which Ga proteins regulate TRPC4 remain unclear. Here, we found that Gai2 regulates TRPC4 activation by direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected cells with the FRET pair. The FRET efficiency between TRPC4J3-CFP and the constitutively active mutant form of Gai2 was nearly 15% and was greater than that observed with wild-type Goti2 (nearly 5%). G$y and the TRPC4 channel showed a FRET efficiency lower than 6%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol increased the FRET efficiency between TRPC4(3-CFP and Gai2(WT)-YFP from 4.7 ± 0.4% (n = 7) to 12.6 ± 1.4% (n = 7). We also found that the TRPC4 channel directly interacts with Gai2, but not with Gaq, when the channel is open. We analyzed the calcium levels in HEK293 cells expressing the channels and Gai2 or Gaq using the calcium indicator YC6.1 (Yellow Cameleon 6.1). In response to the muscarinic agonist carbachol, M2-, Gai2-, and TRPC4-expressing cells showed a prolonged Ca2+ influx compared with cells expressing only M2. Together, these data suggest that Gai2 activates the TRPC4 channel by direct binding, which then induces Ca2+ entry.