The CaMS affects the membrane localization of NCX1 splice variants.

<p>A. HEK293T cells expressing NCX1.1 or NCX1.3, with or without the CaMS (ΔCaMS), were permeabilized and stained with an antibody against the V5 epitope (Green) to label the exchanger. Phalloidin (Red) and DAPI (Blue) were used to visualize actin filaments and nuclear DNA, respectively. Images were obtained on a Leica SP5 confocal microscope. The intensity profiles plotted on the right indicate the red and green fluorescence intensity along the arrows in the corresponding merged images. B. Immunostaining of non-permeabilized HEK293T cells. Left, cells co-expressing NCX1 splice variants (Red) and GFP-GPI (Green). Right, PDM image of the boxed region shown to the left. The grey scale in each image represents the PDM value in each pixel. Scale bar: 10 μm</p

Each of the conserved a.a. residues in the NCX1.1 CaMS has differential effects on exchange activity.

<p>The conserved 1^st, 5^th, 8^th, and 14^th a.a. residues of the CaMS in NCX1.1 was independently mutated from F, V, L, and L, to A (F1A), A (V5A), D (L8D), and D (L14D), respectively. We measured the reverse-mode exchange activity of HEK293T cells expressing these constructs with or without co-expression of CaM or CaM₁₂₃₄. A. Merged images of cells expressing various NCX1.1 mutants stained with phalloidin (Red), DAPI (Blue), and an antibody against the V5 epitope tag (Green) to visualize actin filaments, nuclear DNA, and the exchanger, respectively. Scale bar: 10 μm. B. Representative [Ca²⁺]_i responses from single HEK293T cells expressing various constructs. The line under each trace indicates the period of NMG perfusion. C. Average [Ca²⁺]_i elevations in cells expressing the various NCX1.1 mutants with or without co-expression of CaM or CaM₁₂₃₄. The digits in each column indicate the number of cells in each group. Data are the mean ± SEM pooled from three different batches of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (*: <i>p</i> < 0.05, ***: <i>p</i> < 0.001 when compared with the corresponding group expressing only the NCX1.1 mutant).</p

Schematic illustration of NCX1.

<p>A. Predicted secondary structure of NCX1. NCX has 10 transmembrane segments and the intracellular loop between the 5^th and 6^th transmembrane segments contains several modulatory regions including the exchanger inhibitory peptide (XIP), 2 α2-repeats (shaded regions), Ca²⁺ binding domains (CBD1 and CBD2), an alternative splicing site (AS), and the predicted CaM binding segment (CaMS). B. Sequence alignment of the CaM binding motif. The alignment includes the conserved 1-5-8-14 CaM binding sequences identified in human calcineurin (a.a. 405–424), human plasma membrane Ca²⁺-ATPase 4b (a.a. 1089–1108), human L-type Ca²⁺ channel (a.a. 1601–1620), and human small conductance Ca²⁺-activated K⁺ channel (a.a. 424–443), bovine/human NCX1.1 (a.a. 716–735), human NCX2 (a.a. 666–685), and human NCX3 (a.a. 663–681). Arrows indicates the conserved residues. C. The bovine NCX1 constructs. Empty box indicates the XIP (a.a. 216–270) and boxes filled with different patterns indicate the AS region with different exons. The filled circle indicates the putative CaMS and the empty circle at the C-terminus represents the V5 epitope tag. The number indicates the a.a residue in the NCX1.1. D. Immunostaining of NCX1.1 and NCX1.3 in non-permeabilized cells. HEK293T cells expressing NCX1.1 and NCX1.3 were stained with an antibody against the V5 epitope tagged at the C-terminus of these splice variants. Images were obtained using a Leica SP5 confocal microscope. Scale bar: 10 μm</p

Deletion of exon A/B attenuates exchange activity.

<p>To activate the rNCX activity in HEK293T cells expressing NCX1.4 or NCX1D, we treated them with ouabain and then perfused with NMG buffer. The [Ca²⁺]_i was calibrated based on the changes in fura-2 fluorescence intensities. A. Localization of NCX1.4 and NCX1D. Cells expressing NCX1.4 (upper row) or NCX1D (lower row) were stained with V5 antibody (Green), phalloidin (Red), and DAPI (Blue) to visualize the exchanger, actin filaments, and nuclear DNA, respectively. The intensity profiles plotted on the right indicate intensity of the red and green fluorescence signals along the arrows indicated in the corresponding merged images. Scale bar: 10 μm. B. Representative [Ca²⁺]_i response traces from cells expressing NCX1.4 or NCX1D. The lines under each trace indicate the period of NMG perfusion. C. Average [Ca²⁺]_i changes in cells expressing NCX1.4 and NCX1D with co-expression of CaM or CaM₁₂₃₄. Data presented are the mean ± SEM pooled from three different batches of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (*: <i>p</i> < 0.05, ***: <i>p</i> < 0.001 compared with the group without co-expression).</p

CaM interacts with the NCX1 cytosolic loop.

<p>Purified GST-CaM or GST-CaM₁₂₃₄ was used as bait to pull down intracellular loops subcloned from NCX1.1 or NCX1.3 with (A. NCX1.1CL: a.a. 288–805 and B. NCX1.3CL: a.a. 288–769) or without the CaMS (C. NCX1.1CLΔCaMS and D. NCX1.3CLΔCaMS). During some of the binding reaction, Ca²⁺ (2 mM) was included in the buffer and it was omitted in other reactions. The interacting proteins were verified by Western blots with antibodies against the GST or V5 epitope. M indicates the m.w marker lane.</p

Deletion of the CaMS reduces exchange activity.

<p>HEK293T cells expressing NCX1 and mutants were treated with ouabain to elevate the intracellular Na⁺ concentration. Cells were then locally perfused with NMG buffer for 1 min to induce reverse-mode exchange activity. The [Ca²⁺]_i was calibrated based on changes in fura-2 fluorescence intensities. A. Representative [Ca²⁺]_i traces in single cells expressing different constructs. The black lines under each trace indicate the period of NMG perfusion. Cells transfected with a plasmid missing the exchanger were used as a control. B. The average elevation of [Ca²⁺]_i in cells expressing wild-type NCX1.1 (WT, empty columns) and NCX1.1ΔCaMS (ΔCaMS, filled columns) with CaM or CaM₁₂₃₄ co-expression. C. Average [Ca²⁺]_i responses in cells expressing NCX1.3 WT (empty columns) and NCX1.1ΔCaMS (filled columns) with CaM or CaM₁₂₃₄ co-expression. The digits in each column indicate the sample number. Data are the mean ± SEM pooled from three different sets of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (*: <i>p</i> < 0.05, ***: <i>p</i> < 0.001 compared with WT).</p

Mutations in the conserved a.a. residues of the NCX1.3 CaMS affect exchange activity.

<p>The conserved 1^st, 5^th, 8^th, and 14^th a.a. residues of the CaMS of NCX1.3 was independently mutated from F, V, L, and L, to A (F1A), A (V5A), D (L8D), and D (L14D), respectively. We measured the reverse-mode exchange activity of HEK293T cells expressing constructs with or without co-expression of CaM or CaM₁₂₃₄. A. Merged images of cells expressing various NCX1.1 mutants stained with phalloidin (Red), DAPI (Blue), and an antibody against the V5 epitope tag (Green) to visualize actin filaments, nuclear DNA, and the exchanger, respectively. Scale bar: 10 μm. B. The [Ca²⁺]_i responses of single HEK293T cells expressing various constructs. The black lines under each trace indicate the period of NMG perfusion. C. Average [Ca²⁺]_i elevations in cells expressing various NCX1.3 mutants with or without co-expression of CaM or CaM₁₂₃₄. The digits in each column indicate the number of cells in each group. Data shown are the mean ± SEM pooled from three different batches of cells and analyzed by a one-way ANOVA with Fisher's post hoc test (***: <i>p</i> < 0.001 when compared with the corresponding group expressing only the NCX1.3 mutant).</p

Data files used in the paper

A zip file including data sets and input files for various analysis methods

mtDNA Haplotype Sequence Alignment

Swamp type and river type haplotype alignments. These data were used to generate Fig.S1

input file for skyline plot anlaysis

This is an example input file for skyline plot ananlysi