Plasma Membrane Ca²⁺-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca²⁺ Elevations

<div><p>Plasma membrane Ca²⁺-ATPase (PMCA) by extruding Ca²⁺ outside the cell, actively participates in the regulation of intracellular Ca²⁺ concentration. Acting as Ca²⁺/H⁺ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca²⁺ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pH_mito and pH_cyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca²⁺ clearance and partially attenuated cellular acidification during KCl-stimulated Ca²⁺ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca²⁺ overload. Cyclosporin and bongkrekic acid prevented Ψ_m loss suggesting the involvement of Ca²⁺-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.</p></div

The effect of 59ΔΨ_m depolarizations.

<p>ΔΨ_m changes were measured 10 min after first KCl stimulation, 10 min after first KCl removal (first recovery phase), 10 min after second KCl stimulation and 10 min after second KCl removal (second recovery phase). The level of TMRE fluorescence in resting conditions (5 mM KCl) in each line was taken as 100% (dotted line). Cyclosporin (1 µM), bongkrekic acid (10 µM) or FK-506 (10 µM) were added 1 h before first KCl stimulation. * P<0.05, KCl stimulated vs. resting cells; ^# P<0.05, inhibitor treated cells vs. non-treated.</p

Single cell characterization of cellular pH in steady-state conditions.

<p>Average resting pH_mito and pH_cyto measured by simultaneous imaging of mitoSypHer and SNARF fluorescence, respectively. * P<0.05, pH_mito vs. pH_cyto within each line; ^# P<0.05, PMCA-deficient lines vs. control cells.</p

The relative contribution of ΔΨ_m and ΔΨ_p to TMRE fluorescence.

<p>(A) The effect of 10 min preincubation with FCCP (1 µM) or oligomycin (6 µM) on TMRE or DiSBAC₂ fluorescence assessed by flow cytometry in 10⁴ cells. The fluorescence level in non-treated cells was taken as 100%. * P<0.05, PMCA-deficient lines vs. control cells within inhibitor treated or non-treated groups; ^# P<0.05, inhibitor treated cells vs. non-treated. (B) The dependence of increased TMRE on ΔΨ_p. Before experiment, the medium was exchanged for Ca²⁺-free buffer (20 mM HEPES, pH 7.4, 2 mM CaCl₂, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM glucose) containing either 5 mM (low K⁺) or 59 mM (high K⁺) KCl, in which cells were incubated for 5 min before the addition of TMRE. After 10 min loading period, cellular TMRE fluorescence was acquired. Data are presented in median/quartiles and represent average values from 10⁴ cells. *P<0.05, PMCA-deficient cells vs. control. (C) Real-time PCR analysis of mitochondrial biogenesis markers (Tfam, Nrf-1 and Pgc-1α) and mitochondrially encoded subunits I and III of cytochrome c oxidase (CcO). The expression level of each gene in control line was taken as 1 (dotted line). The relative fold change after normalization to Gapdh expression is shown. * P<0.05, PMCA-deficient lines vs. control cells. (D) Evaluation of mitochondrial mass with MitoTracker Green TM in fixed cells using TCS S5 confocal microscope. The average fluorescence from n = 9, n = 11, n = 14 cells for C, _2, _3 lines, respectively, was measured with microscope accompanying software. Scale bar 10 µm. FIU – fluorescence intensity units.</p

The response of mitochondrial pH to the inhibition of an efflux and the release component of (Ca²⁺)_c elevations.

<p>(A) The average effect of extracellular Ca²⁺ removal and its subsequent restitution on (Ca²⁺)_c and pH_mito changes in n = 18, n = 12, n = 21 cells for C, _2, _3 lines respectively, pretreated for 20 min with thapsigargin and 2-APB. The insets show SERCA-independent (Ca²⁺)_c clearance (upper) and pH_mito recovery (lower). (B) The average effect of cadmium (VDCCs inhibitor) in n = 29, n = 19, n = 22 cells for C, _2, _3 lines respectively, on KCl-evoked (Ca²⁺)_c influx and concomitant pH_mito changes. (C) The average effect of 5 mM La³⁺ in n = 16 cells for C, _2, _3 lines showing delay in (Ca²⁺)_c clearance and pH_mito alkalization under low extracellular Na⁺. (D) The average effect of extracellular alkaline pH (9.0) followed by pH return to 7.4 in n = 20, n = 12, n = 20 cells for C, _2, _3 lines respectively, on (Ca²⁺)_c elevations and pH_mito changes.</p

Changes in (Ca²⁺)_c and ΔpH are reproduced in transiently transfected cells.

<p>(A) Immunodetection of PMCA2 or PMCA3 in PC12 cells transiently transfected with phosphothioate oligodeoxynucleotide probes. (B) Densitometric analysis of PMCAs showing ∼75% knock-down of target genes. The results are presented as arbitrary units (AU) obtained after normalization to endogenous GAPDH level. The dotted line shows the value for untransfected cells (control cells). * P<0.05 PMCA-downregulated cells vs. control cells. (C) The effect of transient PMCAs silencing on (Ca²⁺)_c in n = 17 cells for each line without or (D) with the presence of thapsigargin and 2-APB in n = 20 cells for each line. (E) corresponding changes in ΔpH without or (F) with the presence of thapsigargin and 2-APB.</p

Ca²⁺- and PMCA-dependent mitochondrial acidification.

<p>Cells expressing mitoSypHer were loaded with 10 µM Fura-2 for 1 h and simultaneous changes in Fura-2/mitoSypHer fluorescence were recorded in single cells. The traces show the mean response of (Ca²⁺)_c (upper panel) and pH_mito (lower panel) in n = 11 cells for C, _2, _3 lines following repetitive 59 mM KCl stimulation and recovery.</p

Changes in the mitochondrial pH gradient (ΔpH) during and after 59 mM KCl stimulation.

<p>(A) Simultaneous recordings of pH_cyto and pH_mito in cells expressing mitoSypHer loaded with 10 µM SNARF for 40 min. The cells were imaged in resting conditions (5 mM KCl) and following repetitive stimulation with KCl (59 mM). For each measurement, ΔpH was estimated as pH_mito−pH_cyto. (B) average changes in pH_cyto (i), pH_mito (ii), ΔpH (iii) during each KCl stimulation and in ΔpH after KCl removal (iv) from n = 24, n = 26, n = 23 cells for C, _2, _3 lines respectively. * P<0.05, PMCA-deficient lines vs. control cells.</p

Contribution of electron transport chain (ETC) to mitochondrial H⁺ fluxes.

<p>The traces showing the effect of rotenone (A), KCN (B) or oligomycin (C) on 59 mM KCl-evoked pH_mito responses in n = 10, n = 13, n = 15 for C, _2, _3 lines, respectively. The column graphs show the drug effects on pH_mito loss. * P<0.05 drug treated vs. untreated.</p

TMRE fluorescence decay upon FCCP treatment and the effect on (Ca²⁺)_c.

<p>(A) The representative micrographs showing a decay in TMRE fluorescence in a single cell after 30 s depolarization with 1 µM FCCP. Scale bar 10 µm. (B) Representative traces showing average FCCP-induced Fura-2 fluorescence increase due to release of mitochondrial Ca²⁺ in n = 15, n = 20, n = 19 cells for C, _2, _3 lines, respectively. (C) Representative traces of average Fura-2 fluorescence showing lack of 6 µM oligomycin effect on (Ca²⁺)_c in 14 cells.</p