Fig 2: Pls bound to peripheric juxtamembrane sides.(A) One PI molecule bound to each NHE3 subunit is displayed as spheres with colored atoms, and lipid molecules at the dimerization interface are shown in the same way, with carbon backbone in gray. (B) The electrostatic surface around PI, which is displayed as yellow stick. (C) Interactions between PI and the surrounding residues from TM4 and TM11. (D) Cytoplasmic-viewed PI and anchored HC2. The core and dimerization domain of NHE3 protomer are displayed as a blue solid surface and cylindrical cartoon, while HC2, TM7, and TM9 are displayed as a rainbow cartoon. Residues involving in HC2 stabilization together with PI are displayed as sticks and indicated. (E to G) NHE3 activity was assessed by measuring cytoplasmic pH following an NH4+-induced acidification of AP-1 cells transfected with the various PI-mutant NHE3 constructs. Recovery of pH was initiated by addition of an isotonic Na+-rich solution (indicated by an arrow). For comparison, the pH recovery profile of wild-type NHE3-CHP1 presented in Fig. 2D is reproduced as a dashed line. Tracings are average values (means ± SEM) from three to six cells and representative of two independent experiments. (H) NHE3 activity was defined as the initial linear rate of pH recovery as a function of time [ΔpH/Δt (min−1)] upon reintroduction of a Na+-rich solution. Values represent the means ± SD (n = 9 to 11 cells from two independent experiments). Significance from wild-type NHE3-CHP1 was determined by one-way ANOVA with post hoc Tukey test; ***P < 0.001.

Fig 3: C-terminal motif blocks inward cavity as a plug.(A) Overall view of the HLH motif. Cavity-forming helices from the TMD, i.e., TMs 2, 3, 5, 6, 10, and 12 and the electrostatic inner surface of the inward cavity in NHE3-CHP1 are displayed. Each plug is colored in magenta, and its helices are shown as cylinders. Interactions between plug helices, CHP1, and HC1 (B) and between the plug loop and cavity-forming helices (C) are shown individually. Plugs are colored in magenta, and putative interactive residues are displayed as sticks. (D to F) NHE3 activity was assessed by measuring the cytoplasmic pH following an NH4+-induced acidification of AP-1 cells transfected with various HLH motif mutant NHE3ChFP constructs. Recovery of pH was initiated by addition of an isotonic Na+-rich solution (indicated by an arrow). For comparison, the pH recovery profile of wild-type NHE3-CHP1 presented in Fig. 2D is reproduced as a dashed line. Tracings are average values (means ± SEM) from three to five cells and representative of at least two independent experiments. (G) NHE3 activity was defined as the initial linear rate of pH recovery as a function of time [ΔpH/Δt (min−1)] upon reintroduction of a Na+-rich solution. Values represent the means ± SD (n = 7 to 13 cells from at least two independent experiments). Significance from wild-type NHE3-CHP1 was determined by one-way analysis of variance (ANOVA) with post hoc Tukey test; ***P < 0.001.

Fig 4: Regulation of the NHE3 by CHP1.(A) Biochemical assessment of NHE3 protein expression and membrane trafficking. Chinese hamster ovary AP-1 cells were transiently transfected with a single plasmid encoding either mCherry fluorescent protein [ChFP; no transporter (NT)], NHE3, or NHE3-CHP. Total cell lysates (TCLs; 40 μg of protein per lane) and plasma membrane (PM) fractions (1 mg of protein per lane) were analyzed by Western blotting using mouse monoclonal antibodies to detect ChFP (NHE3), CHP1, β-tubulin, and ATP1A1 (Na+/K+-ATPase 1 subunit). β-Tubulin and ATP1A1 served as loading controls for TCL and PM fractions, respectively. (B) Densitometric quantification of the surface abundance of NHE3 in the absence or presence of CHP1 was normalized to the total cellular expression of NHE3 and β-tubulin. Data are shown as means ± SD of three separate experiments. Statistical significance was assessed using the unpaired two-sample t test; *P < 0.05. (C) Immunofluorescence of AP-1 cells transiently transfected with NHE3 or NHE3-CHP1. Nuclei were labeled with DNA dye 4′,6-diamidino-2-phenylindole. Images show each channel individually, with merged images of the NHE3 and Tf-Alexa488 channels. Scale bars, 10 μm. Images are representative of two independent experiments. (D) NHE3 activity was assessed by measuring cytoplasmic pH recovery following an NH4+-induced acid load in AP-1 cells transfected with ChFP (NT), NHE3, or NHE3-CHP1. Recovery of pH was initiated by addition of an isotonic Na+-rich solution (indicated by an arrow). Tracings are average values (means ± SEM) from three to five cells and representative of at least two independent experiments.