Activation mechanisms of TRPC4 channel: direct interaction with Gαi2, membrane targeting, and tetrameric structure formation

Abstract

Canonical Transient Receptor Potential (TRPC) channels are Ca2+-permeable nonselective cation channels that are activated by a wide variety of stimuli, including a family of G protein-coupled receptors (GPCRs). It is still intriguing whether TRPC4 channel is receptor-operated channel (ROC) or store-operated channel (SOC), and recent studies claim that TRPC4 is ROC rather than SOC. However, molecular mechanisms by which Gα proteins participate to regulate TRPC4 remain unclear. Furthermore, the mechanism underlying the activation of TRPC4 channels is not clearly understood. The first aim of this study is to evaluate function of Gαi2 to regulate TRPC4 activation via direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence (Förster) resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected HEK293 cells with the FRET pair. The FRET efficiency between TRPC4β and the constitutively active mutant form of Gαi2 was nearly 14% and was greater than that observed with wild type (WT) Gαi2 (nearly 5%). Gβγ and the TRPC4 channel demonstrated a FRET efficiency lower than 4%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol (CCh) increased the FRET efficiency between TRPC4β and Gαi2 from 5.2 ± 1.2% (n = 7) to 12.5 ± 1.4% (n = 7). We also found that the TRPC4 channel directly interacts with Gαi2 but not with Gαq when the channel is open. Since TRPC4 channel is a membrane protein, it functions when localized at the membrane to be fully activated. Thus, our second aim of this study is to find membrane targeting domain of TRPC4. To identify the regulating region of the channel trafficking to the membrane, we generated deletion mutants of the TRPC4 channel. We determined that when either region that was downstream of the 20 amino acids of the N-terminus or the 700-730 domain was deleted, the mutants were retained in the endoplasmic reticulum. By coexpression of the TRPC4 with deletion mutants, we found that the 23-29 domain of the N-terminus regulate the membrane trafficking. We inferred the candidate proteins that regulate or interact with the 23-29 domain of TRPC4. TRPC4 channel functions when folded to tetrameric structure which consists of 4 subunits. In this study, we evaluate that the downstream region of 99th amino acid in the N-terminus and upstream region of 730th amino acid in the C-terminus are involved in the assembly of TRPC4 tetramers. Meaning, TRPC4 ion channel opens by the activation of Gαi2 by stimulus of GPCR. Also, for this ion channel to function properly, it requires the manifestation of cell membranes and formation of tetrameric structure, and the domain which induces the manifestation of the cell membrane is 23-30 domain amino acid of N-terminus. Lastly, the domain required to create a functioning tetrameric ion channel exists in both the N-terminus and the C-terminus.