Figures and data
PIP2 enhances HsNCX1 activity.
(A) Representative outward currents recorded from oocytes expressing the human NCX1 before and after application of long-chain brain PIP2. Currents were activated by replacing cytosolic Cs+ with Na+. Application of 10 µM brain PIP2 enhanced HsNCX1 current and abolished the Na+-dependent inactivation irreversibly. Perfusion time of PIP2 is indicated above traces while lines below traces indicate solution exchange. Arrows mark the peak and steady currents used to measure the fold of increase upon PIP2 application. The fold of current increase was calculated by comparing the peak or steady-state current before and after PIP2 application.
(B) Representative outward currents recorded before and after application of short-chain PIP2 diC8 (10 µM). PIP2 diC8 was perfused from the cytosolic side before HsNCX1 activation (in the presence of Cs+ for 30 s) and during transport (in the presence of Na+). Both peak and steady-state currents of HsNCX1 are enhanced by PIP2 diC8 and the effect is reversible. The Na+-dependent inactivation remains in the presence of PIP2 diC8.
PIP2 binding in NCX1.
(A) Structure of the TM and β-hub regions of PIP2 diC8-bound NCX1 with a zoomed-in view of the lipid binding site.
(B) Structural comparison between apo (grey) and PIP2 diC8-bound (color) NCX1.
(C) Zoomed-in view of the structural comparison (boxed area in (B)). The two major conformational changes occur in the boxed regions with the two areas SEA0400 binding site and the schematic diagram detailing the interactions between NCX1 residues and SEA0400.
(D) Zoomed-in views of the two conformational changes between apo (left in grey) and PIP2 diC8-bound (right in color) state. Top: conformational change 1 at the C-terminus of TM5. Bottom: conformational change 2 at the interface between XIP and CBD2.
Mutagenesis at the PIP2 binding.
(A) Representative HsNCX1 currents recorded of single mutants before and after perfusion of 10 µM brain PIP2 to the cytosolic side of the patch.
(B&C) Summary graphs demonstrating the effects of PIP2 on the enhancement of peak (B) and steady-state currents (C). Potentiation (fold of increase) was measured by comparing the current magnitude before and after PIP2 application. Mutants R167A, R220A, and K225A showed decreased response to PIP2, while the double mutant R220A/K225A response to PIP2 at steady state decreased by ∼70% (Fold of increase WT=8.9±1.6, n=10 vs R220A/K225A=2.4±0.5, n=7). Data points are mean ± s.e.m. (* p <0.1).
(D) The extent of Na+-dependent inactivation was measured as the ratio between steady state and peak currents (fractional activity) and values for WT and the indicated mutants are shown. Mutants R220A and R220A/K225A displayed significantly higher fractional activity values when compared to WT, indicating that the Na+-dependent inactivation was less pronounced in these mutant exchangers.
SEA0400 binding in NCX1.
(A) Overall structure of human NCX1 in complex with SEA0400 obtained in high Na+ and low Ca2+ conditions. Yellow spheres represent the bound Ca2+ in CBD1 and XIP.
(B) Cartoon representation of the TM domain and β-hub of the complex with surface-rendered view of the fenestration in the middle of the membrane. The β-hub is assembled by β-hairpin (β1 and β2) and XIP (β3 and β4).
(C) Zoomed-in view of SEA0400 binding site and the schematic diagram detailing the interactions between NCX1 residues and SEA0400.
Structural mechanism of SEA0400 inhibition.
(A) Structural comparison at the core part of the TM domain between the SEA0400-bound, inward-facing HsNCX1 and the outward-facing NCX_Mj (PDB 3V5U). Red arrows mark the sliding movement of TMs 1 and 6 and the bending of TM2ab from inward to outward conformation.
(B) Surface-rendered views of the SEA0400-binding pocket sealed off from the cytosolic side by E244 from XIP in the inactivated state.
(C) Removal of XIP would generate a cytosolic portal that facilitates the release of SEA0400.
Cryo-EM data processing of HsNCX1 in the presence of long-chain porcine brain PIP2.
The structural model of Ca2+-activated HsNCX1 from a previous study (PDB 8SGT) was directly fitted into the low-resolution EM-map (∼11.5 Å). The Fab fragment from a monoclonal antibody against NCX1 was used as a fiducial marker to facilitate the single-particle alignment.
Structure determination of HsNCX1-PIP2 diC8 complex.
(A) Cryo-EM data processing of HsNCX1 in complex with short-chain PIP2 diC8. The Fab fragment from a monoclonal antibody against NCX1 was used as a fiducial marker to facilitate the single-particle alignment.
(B) Zoomed-in view of density map of the bound PIP2 diC8 contoured at the threshold level of 0.43 using ChimeraX.
(C) The Fourier shell correlation (FSC) curves for cross-validation between the maps and the models.
Structure determination of HsNCX1-SEA0400 complex.
(A) Cryo-EM data processing scheme of HsNCX1 in complex with SEA0400 inhibitor. The Fab fragment from a monoclonal antibody against NCX1 was used as a fiducial marker to facilitate the single-particle alignment.
(B) Zoomed-in view of density map of the bound SEA0400 inhibitor contoured at the threshold level of 0.52 using ChimeraX.
(C) The Fourier shell correlation (FSC) curves for cross-validation between the maps and the models.
Structural comparison between the apo (grey) and SEA0400-bound (color) HsNCX1.
