Fig. 1: PI(4,5)P2 depletion inhibits human PAC channels.
a Voltage-dependent currents of hPAC channels in extracellular pH 7.4 or 4.6 solutions in wild-type (WT) HEK293T cells expressing PAC channels endogenously and PAC CRISPR knockout KO3 cells. b (left) Representative confocal images of KO3 cells expressing hPAC1-GFP (top) or hPAC2-GFP (bottom) with the plasma membrane marker Lyn-mCherry (Lyn-mCh). The yellow arrow indicates the location and line scanning direction in merged images. Scale bar: 10 μm. (right) Line-scan analyzes of Lyn-mCh and hPAC1-GFP (top) or hPAC2-GFP (bottom). *, peak cytosolic fluorescence intensity. Each experiment was repeated 3 times independently with similar results. c Current density–voltage relationship for hPACs at various pH conditions. n = 5 for pH 7.4, 6.3, 5.3, 5.0; n = 10 for pH 5.5; n = 7 for pH 4.6 in hPAC1; n = 2 for pH 7.4; n = 5 for pH 6.3, 5.5; n = 3 for pH 5.3, 5.0; n = 4 for pH 4.6 in hPAC2. Data are mean ± SEM. d (left) Scheme for PI(4,5)P2 depletion by Dr-VSP. (right) ΔFRETr changes in control (top) or cells transfected with Dr-VSP (bottom) during +100-mV/10-s depolarization pulse. Cells were transfected with the PI(4,5)P2 FRET probes, PHPLCδ1-CFP and PHPLCδ1-YFP. e pH 5.5-induced PAC current traces during test pulse in KO3 cells (top) and transfected with hPAC1 (middle) or hPAC2 (bottom), without (control) or with Dr-VSP. f Current density measured by analyzing the steady-state current at the end of each +100-mV depolarization step in (e). KO3: n = 8; n = 8. hPAC1: n = 6; n = 7. hPAC2: n = 9; n = 6 for −Dr-VSP and +Dr-VSP, respectively. Dots indicate the individual data points for each cell. Data are mean ± SEM. g Average percent inhibition of current densities by PI(4,5)P2 depletion in different pH solutions. h hPAC1 current recovery from Dr-VSP-induced inhibition in cells intracellularly perfused with (top) or without (bottom) ATP. PAC currents were measured at the indicated times after a + 120-mV/10-s depolarization pulse. Dotted lines indicate zero, and dashed lines indicate PAC current amplitude (control) before the depolarizing step. i Average current recovery with (open) or without (filled) of intracellular ATP (n = 6, +ATP; n = 4, -ATP). Data are mean ± SEM. Source data are provided as Source Data file.
