Functional Characterization of Na⁺/H⁺ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Abstract

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na + /H + exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane.The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na + /H + exchangers used for the selection. Video LinkThe video component of this article can be found at

Intracellular pH Control by Membrane Transport in Mammalian Cells. Insights Into the Selective Advantages of Functional Redundancy

“…In this case pH measurements just turn to be fluorescence spectroscopy measurements. Different setups can be used, from videomicroscopes equipped with camera and illumination systems (Milosavljevic et al, 2015) to fluorimeters or microplate readers equipped with fluorescence. Using fluorescence, pH values can be in principle deduced from a modified Henderson-Hasselbalch equation adapted to fluorescence, or fluorescence ratio when ratiometric probes are used.…”

The cleaved FAS ligand activates the Na+/H+ exchanger NHE1 through Akt/ROCK1 to stimulate cell motility

“…Measurements were performed by 1-minute incubation in the uptake medium at room temperature supplemented with 3 mmol/L LiCl followed by four rapid rinses in ice-cold PBS as described in37 Cells were solubilized in 1 N nitric acid (trace metal grade, Fisher Scientific), and Li + was measured using atomic absorption spectrometry (Zeeman furnace system, Solaar 969, Thermo Optek). NHE-1 initial rates were calculated as the cariporide (10 μmol/L)–sensitive Li + accumulation per well divided by protein quantities and normalized to maximal values.…”