Journal Article

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

Stepinski, Dariusz, Kowalski, Antoni, Ferenc, Bozena, Zylinska, Ludmila, Boczek, Tomasz, Lisek, Malwina, Wiktorska, Magdalena

2014

Overview

Plasma membrane Ca(2+)-ATPase (PMCA) by extruding Ca(2+) outside the cell, actively participates in the regulation of intracellular Ca(2+) concentration. Acting as Ca(2+)/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 Ca(2+) overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pHmito and pHcyto 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 Ca(2+) clearance and partially attenuated cellular acidification during KCl-stimulated Ca(2+) 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 Ca(2+) overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca(2+)-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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Acidification / Adenosine triphosphatase / Animals / Biology and Life Sciences / Bongkrekic Acid - pharmacology / Ca2+-transporting ATPase / Ca2+/H+-exchanging ATPase / Calcium (intracellular) / Calcium (mitochondrial) / Calcium - metabolism / Calcium influx / Calcium ions / Calcium permeability / Cell Differentiation / Cell Membrane - drug effects / Cell Membrane - metabolism / Chlorides / Cyclosporine - pharmacology / Cytosol - drug effects / Cytosol - metabolism / Electrochemistry / Enzyme Inhibitors - pharmacology / Extrusion / Gene Expression Regulation / Homeostasis / Homeostasis - physiology / Hydrogen ions / Hydrogen-Ion Concentration - drug effects / Intracellular / Ion Transport - drug effects / Isoforms / Localization / Membrane composition / Membrane permeability / Membrane potential / Membrane Potential, Mitochondrial - drug effects / Membrane Potentials - drug effects / Membranes / Metabolism / Metabolites / Mitochondria / Mitochondria - drug effects / Mitochondria - metabolism / Mitochondrial DNA / Mitochondrial Membrane Transport Proteins - antagonists & inhibitors / Mitochondrial Membrane Transport Proteins - genetics / Mitochondrial Membrane Transport Proteins - metabolism / Mitochondrial permeability transition pore / Na+/Ca2+ exchanger / Neurochemistry / Neurons / PC12 Cells / Permeability / pH effects / Pheochromocytoma cells / Phosphorylation / Plasma / Plasma Membrane Calcium-Transporting ATPases - antagonists & inhibitors / Plasma Membrane Calcium-Transporting ATPases - genetics / Plasma Membrane Calcium-Transporting ATPases - metabolism / Potassium chloride / Potassium Chloride - pharmacology / Preservation / Protons / Rats / Rodents / Signal Transduction / Steady state