Abstract
Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gα(q)-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) maintains TRPC4 and TRPC5 channel function, the Gα(q)-PLC pathway inhibits channel activity by depleting PI(4,5)P(2). Here we investigated the difference in PI(4,5)P(2) sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P(2) 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)P(2) 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)P(2) binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P(2) binding sites because the mutations reduced FRET to a PI(4,5)P(2) 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)P(2) affinity for TRPC4 and TRPC5 that changes PI(4,5)P(2) sensitivity.