Fig. 6. NHE1 activity, but not membrane localization, is attenuated by disruption of the NHE1-LID:membrane costructure.

a, b Plasma membrane expression of NHE1 in AP-1 cells (untransfected or stably expressing wt or 4G-NHE1 as indicated), assessed by pull-down of the biotinylated membrane fraction followed by immunoblotting for NHE1. a Representative immunoblots. β-actin was used as a loading control (no signal in the pull-down fraction, as expected). Uncropped blots are available in Supplementary Fig. S12. b Plasma membrane NHE1 expression normalized to that in wt clone 1. Data are quantified from n = 4 biologically independent experiments per clone and shown as mean with S.E.M error bars and all individual data points. The membrane expression of 4G-NHE1 did not differ significantly from that of NHE1 (pooled data from both clones for each variant, two-tailed, non-paired Student’s t-test, p > 0.05). c Localization of wt- and 4G-NHE1 in AP-1 cells. Cells were fixed and stained with antibody against NHE1 (red), rhodamine-conjugated phalloidin to visualize F-actin (green), and DAPI to visualize nuclei (blue). Arrows in the detail images highlight the membrane localization of NHE1. Data shown are representative of n = 3 biologically independent experiments per condition. d–g To measure wt- and 4G-NHE1 activity, cells were loaded with BCECF-AM, and pH_i monitored using real time fluorescence imaging. d Steady-state pH_i, averaged over the two NHE1 wt (n = 19 biologically independent experiments) and 4 G (n = 16 biologically independent experiments) cell clones. Data are shown as mean with S.E.M. error bars and all individual data points. e Representative examples of pH_i traces. The black arrow indicates the point of removal of NH₄Cl. f pH_i recovery rates for the NHE1 wt (n = 20 biologically independent experiments) and 4 G (n = 15 biologically independent experiments) variant, calculated from the initial linear part of the pH_i traces after maximal acidification, as in e. Data are shown as mean with S.E.M. error bars. g From traces as in (E), pH_i recovery rates as a function of pH_i was calculated by fitting the recovery rates over the entire recovery period. Data are shown as mean with S.E.M. error bars. Data in F and G were corrected for relative cell surface expression (data from Panel B) to ensure that the pH_i recovery represents the capacity of the membrane-expressed fraction of NHE1. **p = 0.0024 (panel D) and 0.0016 (panel F) and compared to wt, two-tailed, non-paired Student’s t-test using GraphPad Prism 8.4.1 software and assuming normal distribution. Source files for Fig. 6b,d–g available as supplementary data.