Fig 2: The NHE1-LID binds different lipids with induction of helical structures.a Position of individual lipids on the dot blot membrane. b Lipid binding profile of the CHP1/NHE1503-595 and CHP1/NHE1503-698 at pH 7.4 and 8.4. c Variants of NHE1 used to test lipid binding specificity. d Effect of various mutations on CHP1/NHE1503-595 lipid binding. e Far-UV CD spectra of NHE1-LID539-593 in DHPC detergent (color) and in DMPC:DHPC bicelles (dashed color). The CD spectrum of NHE1-LID539-593 in water is shown in black. f Far-UV CD spectra of the NHE1-LID in anionic bicelles consisting of DMPG:DMPC:DHPC (dashed color) and in 2% LPPG (color). The CD spectrum of NHE1-LID539-593 in water is shown in black. g 15N,1H-HSQC spectrum of NHE1-LID539-593 in 2% LPPG, 320.15 K. h SCSs of Ca of the NHE1-LID in 2% LPPG with two highly populated helices, H1 and H2, indicated below the sequence.

Fig 3: Ion influx at protrusion mediated by sodium hydrogen exchanger-1 and transient receptor potential vanilloid–4 ion channels.(A) Intracellular sodium ion imaging in cell protrusion (white dashed box). (B) Distribution profile of intracellular sodium ions along protrusion (n > 45 single cells). (C) Intracellular calcium ion imaging in cell protrusion (white dashed box). (D) Distribution profile of intracellular calcium ions along protrusion (n > 45 single cells). (E) Time-lapse images of a cell merged with heatmaps of calcium ion concentration. Scale bar, 5 µm. (F) Evolution intracellular calcium ion concentration at end of protrusion (n > 45 single cells). (G and H) Representative images and profiles of intracellular sodium ions with sodium hydrogen exchanger-1 (NHE-1) inhibition (n > 45 single cells). (I and J) Representative images and profiles of intracellular calcium ions with transient receptor potential vanilloid–4 (TRPV4) inhibition (n > 45 single cells). (K) Western blot analysis of NHE-1 and TRPV4 protein expression in MSCs with siRNA of NHE-1 (siNHE), TRPV4 (siTRPV4), or control (siCNTR). (L to N) Representative 3D track reconstructions (L), track lengths (M), and mean speeds (N) for migration of MSCs (n > 200 single cells). Scale bars are 10 µm unless otherwise indicated. ****P < 0.0001 by one-way ANOVA test. All data are shown as means ± SEM, including profile graphs.

Fig 4: NHE1 T779 is rapidly and specifically dephosphorylated by CN. a Overlay of extracts from the 2D [1H,15N] HSQC spectra of ERK2-phosphorylated NHE1ct before (red), and after the addition of CN (black). Addition of CN resulted in disappearance of peaks for the phosphorylated state of T779 and reappearance of peaks reporting on the unphosphorylated state of T779 (V777, dashed circle). The peaks corresponding to the five SP phosphorylation sites were not affected. b Time course of in vitro NHE1ct dephosphorylation by CN following the peaks from the six ERK2 phosphorylated residues. Cartoon of NHE1ct (colored as in Fig. 1c), with the symbols of the phosphorylation sites corresponding to the dephosphorylation time-course curves shown below. c Top: Representative immunofluorescence (IF) images of the six variant PS120 cell lines stained for NHE1 (red) and DAPI (blue) to evaluate NHE1 expression and localization. Images were taken at ×60 magnification on an Olympus Cell Vivo microscope. Scale bars represent 20 µm (n = 2). Bottom: Representative line scans showing the membrane localization of each variant. (For overview images confirming clonality and cell surface biotinylation to confirm membrane localization see Supplementary Fig. 7.) d Representative Western blot of the six variant PS120 cell lines blotted for NHE1. The lower band corresponds to immature unglycosylated NHE1 and the upper band to glycosylated NHE1; p150 was used as loading control. e Total NHE1 expression was quantified using ImageJ and normalized for each variant to the loading control and then to the expression of the WT cell line. One-way ANOVA with Dunnet’s post-test (n = 5 biologically independent experiments for all cell lines except 779, where n = 3). f Representative traces of BCECF-AM fluorescence converted to pHi for the six variant PS120 cell lines showed over the time course of addition of Na+-free solution and Ringer solution. The recovery rates as ?pH*10–3*s-1 of each cell line are depicted in g. One-way ANOVA with Dunnet’s post-test. (The number of biologically independent experiments were as follows: T779A: 6n; WT, ATAP, AVATAA: 5n; P120, ATA/AVATAA: 4n; 779D: 3n.) * and *** denotes p < 0.05 and p < 0.001, respectively. Error bars represent S.E.M. values. Source data are provided as a Source Data file

Fig 5: Calcium (Ca2+) dynamics affect cell migration speed by regulating ion-channel and F-actin localization at the cell leading edge. Intracellular Ca2+ levels (measured as fluorescence signal from GCamp6m transfected live cells) in different media conditions. Representative GCamp6m fluorescent images in A) DMEM (immediately after addition), B) 1% MC (immediately after addition), and C) 1% MC (long term >3 h incubation). D) Cell speed under various levels of Ca2+ chelation via BAPTA-AM (DMEM DMSO Ctrl N=182; 1% MC DMSO Ctrl N = 411; DMEM 5 µm BAPTA-AM N=135; 1% MC 5 µm BAPTA-AM N=144; DMEM 10 µm BAPTA-AM N = 117; 1% MC 10 µm BAPTA-AM N = 137; DMEM 20 µm BAPTA-AM N=86; 1% MC 20 µm BAPTA-AM N = 127). E) Cell speed upon non-specific inhibition of mechano-sensitive channels via GdCl3 (DMEM Water Ctrl N = 45; 1% MC Water Ctrl N = 102; DMEM 100 µm GdCl3 N=96; 1% MC 100 µm GdCl3 N = 157). F) Cell speed upon inhibition of TRPM7 via FTY (DMEM DMSO Ctrl N=77; 1% MC DMSO Ctrl N = 154; DMEM 2 µm FTY N = 83; 1% MC 2 µm FTY N = 120). G) Cell speed upon treatment with extracellular Ca2+ chelator BAPTA (DMEM DMSO Ctrl N = 162; 1% MC DMSO Ctrl N=152; DMEM 50 µm BAPTA N = 146; 1% MC 50 µm BAPTA N = 144). H) Cell speed upon treatment with 2-APB and CAI (DMEM DMSO Ctrl N = 145; 1% MC DMSO Ctrl N = 149; DMEM 200 µm 2-APB + 40 µm CAI N=169; 1% MC 200 µm 2-APB + 40 µm CAI N = 178). Leading edge intensities of ion-channels and F-actin are reduced under BAPTA-AM treatment as compared to DMSO Control: I) Leading edge intensities of NKCC1 (1% MC DMSO Ctrl N = 132; 1% MC 10 µm BAPTA-AM N = 115), J) Leading edge intensities of NHE1 (1% MC DMSO Ctrl N = 132; 1% MC 10 µm BAPTA-AM N = 171), K) Leading edge intensities of NaK (1% MC DMSO Ctrl N = 133; 1% MC 10 µm BAPTA-AM N = 144), and L) Leading edge intensities of F-actin (1% MC DMSO Ctrl N = 397; 1% MC 10 µm BAPTA-AM N = 430). *p < 0.05, **p < 0.005 and ***p < 0.0005 across all plots. Error bars represent standard deviation of the data.