Fig. 4: Identification of residues critical for hPAC1 PI(4,5)P2 regulation through alanine scanning. | Nature Communications

Fig. 4: Identification of residues critical for hPAC1 PI(4,5)P2 regulation through alanine scanning.

From: The plasma membrane inner leaflet PI(4,5)P2 is essential for the activation of proton-activated chloride channels

Fig. 4

a Sequence of positively charged residues clustering in hPAC1. Purple lines indicate N- and C-terminal sections. Green lines indicate transmembrane domain (TM) 1 and 2 sections. Positively charged residues indicated in blue are selectively mutated to alanine (A). b Representative confocal images of KO3 cells expressing wild-type (WT) or alanine-substituted hPAC1 mutants with the plasma membrane marker Lyn-mCh. Scale bar: 10 μm. Scatter plots in right panels display a 2D intensity histogram between hPAC1-GFP and Lyn-mCh pixels in the confocal image. Each experiment was repeated 3 times independently. c Representative current traces of WT or mutant channels elicited at +100 mV in the control (left) and cells expressing Dr-VSP (right). d Pearson’s correlation coefficients (R) for the colocalization between hPAC1-GFP and Lyn-mCh confocal images. n = 6 for WT; n = 10 for K120A, K390A, K397A, R398A, K401A; n = 9 for K124A, K394A, R396A, K394/R396A/K397A, K402A, K403A. Dots indicate the individual data points for each cell. Data are mean ± SEM. e hPAC1 current densities at +100 mV for control and cells expressing Dr-VSP in pH 5.5. The dashed gray line indicates the standard WT level. n = 6 for WT, K394A, K402A; n = 7 for K120A; n = 4 for K124A; n = 5 for K390A, R396A, K397A, K394/R396A/K397A, R398A, K403A; n = 3 for K401A. Data are mean ± SEM. ns, not significant compared to WT. One-way ANOVA followed by Dunnett’s multiple comparisons test. P values: 0.9993, K120A; 0.1938, K124A; 0.9994, K390A; 0.0009, K394A (***P < 0.001); <0.0001, K396A (****P < 0.0001); 0.0103, K397A (*P < 0.05); <0.0001, K394/R396/K397A (****P < 0.0001); 0.8065, K398A; 0.9938, K401A; 0.9999, R402A; 0.9249, R403A. f Families of hPAC1 current of WT (left) and K394A/R396A/K397A mutant (right) elicited by the voltage steps shown at the top under different extracellular pH condition. g pH-dependent hPAC1 current measured at +100 mV in cells expressing hPAC1 WT (n = 6 for pH 7.4, 5.8, 5.5; n = 5 for pH 6.3; n = 7 for pH 5.3, 5.0) or K394A/R396A/K397A (n = 5 for pH 7.4, 6.3, 5.8, 5.5, 5.3, 5.0). Data are mean ± SEM. Source data are provided as Source Data file.

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