Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels

Abstract

Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gαq-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) maintains TRPC4 and TRPC5 channel function, the Gαq-PLC pathway inhibits channel activity by depleting PI(4,5)P2. Here we investigated the difference in PI(4,5)P2 sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P2 dephosphorylation robustly inhibits TRPC4α, TRPC4β, and TRPC5 homotetramer currents and also TRPC1/4α, TRPC1/4β, and TRPC1/5 heterotetramer currents. Secondly, sensitivity of channels to PI(4,5)P2 dephosphorylation was suggested through the usage of FRET in combination with patch clamping. The sensitivity increased in the sequence TRPC4β < TRPC4α < TRPC5 in homotetramers, whereas when forming heterotetramers with TRPC1, the sensitivity was approximately equal between the channels. Thirdly, we determined putative PI(4,5)P2 binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P2 binding sites because the mutations reduced FRET to a PI(4,5)P2 sensor and reduced the current amplitude. Therefore, one functional TRPC4 has 8 pockets with the two main binding regions; K419, K664/R511, K518, H630. We conclude that TRPC1 channel function as a regulator in setting PI(4,5)P2 affinity for TRPC4 and TRPC5 that changes PI(4,5)P2 sensitivity.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

“…PLC modulation on TRPCs is inseparable with PI(4,5)P 2 , the substrate of PLC. TRPC1/4 and TRPC1/5 channels interact with the PH domain of PLCδ through PI(4,5)P 2 [32]. PI(4,5)P 2 was shown to be essential for TRPC4 activation [3].…”

Structure–Function Relationship and Physiological Roles of Transient Receptor Potential Canonical (TRPC) 4 and 5 Channels

“…A different study was reported which shares the similar context that PIP 2 is necessary to maintain the opening of TRPC4β [37]. In this study a voltage-sensitive phosphatase (VSP) system was employed.…”

“…However, most of all, it was rapid and prominent desensitization of the channels that made dissecting downstream components out of overall effect of the Gq pathway to TRPC channels challenging. With an astonishing consistency, TRPC4 or 5 channels showed prominent desensitization right after strong activation whenever the channels were activated by Gq-protein coupled receptors (GqPCR, e.g., mAChR1, or 3, histamine receptor subtype 1) [19,21,22,[37][38][39][40][41][42][43]59,71,72]. As an alternative, a non-specific G-protein activator such as [GTPγS] i has been used [35,45,46].…”

“…Classically, TRPC4 and 5 channels are known to be activated by G-proteins and their downstream signal transduction pathways, such as Gq-PLC pathway [2,3,21,22,34,35]. Intensive researches have been done on delineating functional relationship among TRPC4, 5 channels and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) [36][37][38][39][40][41][42], intracellular Ca 2+ ([Ca 2+ ] i ) and protein kinase C (PKC) [43,44]. Recently, it was suggested that not only downstream signal transduction pathways but active form of alpha subunits of G-proteins (Gαi or Gαq) per se can activate TRPC4, TRPC5, TRPC1/4, or TRPC1/5 channels as well [38,39,45,46].…”

Regulation of transient receptor potential canonical 4 activity by phospholipase C δ1

“…However, which mechanism makes this difference is still obscure. Many researches demonstrated the effect of downstream molecules on TRPC channels, which includes PLC, phosphatidylinositol 4,5-biphosphate (PIP2) [1][2][3][4] , diacylglycerol (DAG) 5,6 , protein kinase C (PKC) 7 and Ca 2+ . We previously emphasized the self-limiting mechanism of Gαq pathway on channels that Gαq protein solely activates TRPC1/4 and TRPC1/5 channels, and then PIP2 depletion and PKC or Ca 2+ inhibits them 8,9 .…”