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.
Highlights
This article concerns the Transient receptor potential canonical (TRPC) subfamily of ion channels, which includes seven gene members
We began by studying the effects of PI(4,5)P2 depletion on TRPC4 and TRPC5 channel currents
We suspect that the decay of TRPC4 and TRPC5 channel currents is a consequence of the depletion of PI(4,5)P2
Summary
This article concerns the TRPC subfamily of ion channels, which includes seven gene members. Homo- and heteromeric TRPC channels are activated by stimulation of G-protein coupled receptors (GPCR) that induce hydrolysis of PI(4,5) P24,5 and calcium release[6,7]. The TRPC1 subunit may be devoted to a regulatory role in heteromeric channels rather than forming homomeric functional channels. To test how PI(4,5)P2 affects channel activities They include a rapamycin-inducible system[16], a light-dependent optogenetic system[3], and the depolarization mediated voltage-sensitive phosphatase (VSP) system[20]. These methods offer opportunities to explore poorly understood functional roles of PI(4,5)P2 on TRPC4 and TRPC5 channels[7,15,21]. Details in interaction between PI(4,5)P2 and channels are not well established
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