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

Summary

The first part of this article shows how to select mutant cell lines expressing vesicular Na⁺/H⁺ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

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.

Introduction

Most intracellular compartments display an acidic luminal pH, which is a key parameter for maturation, trafficking, recycling of proteins or hormones and neurotransmitters loading. It has been shown that the pH gradient between cytosol and vesicular content is generated by vacuolar H⁺ ATPases¹, coupled to vesicular ClC chloride transporters². Both in knock-out (KO) mice and human patients, the importance of these transporters has been highlighted by the heavy phenotypes caused by mutations in their genes^3-6.

The members of the Sodium-Hydrogen exchangers SLC9A family, also termed NHEs for Na^+<....

Protocol

1. H⁺ Killing Selection

1. Cell lines
   1. Stably transfect NHE-deficient cells (e.g., the CCL39-derived PS120 cell line¹⁶) using any combination of mammalian expression vector and transfection method that will produce efficient transfection yields and selection in a fibroblast cell line.
      NOTE: For many years Calcium Phosphate precipitation¹⁹ was used with good transfection yields. This has been replaced more recently by commercial reagents such as Lipofectamine2000, the transfections being performed following the manufacturer’s instructions.
2. Solutions
   1. Prepare t....

Results

Selection:

The H⁺ killing selection is based on the diffusion of the ammonium weak base, as depicted in Figure 1A. The effect of weak bases and acids diffusion on intracellular pH has been pioneered by Walter Boron and collaborators²². The elegant idea to use this phenomenon to produce a lethal acidification for positive genetic selection was then developed by Jacques Pouysségur¹⁷. Under such a protocol, the intracellular pH drops to about 5.5 .......

Discussion

This protocol describes how to select cells expressing intracellular Na⁺/H⁺ exchangers at the plasma membrane without altering their primary sequence by site-directed mutagenesis. These exchangers can now be characterized.

This method is based on the cellular toxicity of intracellular protons to select cell lines that will express vesicular Na⁺/H⁺ exchangers at the plasma membrane. It might be possible, in principle, to use other cations that may be .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Standard cell culture equipement			Used for H+ killing selection composed of many devices with different catalog numbers
Incubator with CO2	Sanyo	MCO15A
Incubator without CO2	Heraeus instrument	BB6220
Laminar flow hood PSM1200NF	Fisher	52010120
DMEM medium	sigma	D5796
FBS gold	GE Healthcare	A15-151
Penicilin/streptomycin	PAA	P11-010
Trypsin 10x	PAA	L11-003
Atomic Absorption spectrometer with Zeeman furnace system	Thermo Scientific	ICE 3500 GFZ
LiCl	sigma	L4408
Nitric Acid	sigma	438073
Fluorescence videomicroscopy set	Leica		Composed of many devices with different catalog numbers
Inverted automated microscope	Leica	DMI6000B
microscope stand	leica	11888906
		11888911
		11505180
		11888377
incident fluorescence	leica	11888901
		11504166
motor bracket	leica	11888379
		11505234
		11521505
		11522106
LED transmission light	leica	8097321
		8102034
		11521580
motorized plate	leica	11522068
		11531172
		11521734
		11521719
		11888423
		11888424
camera output	leica	11888373
		11507807
		11888393
		11888259
		11888258
		11541510
images acquisition/analysis software	leica	11888375
optics	leica	11506507
		11506243
		11506203
fluorescence Xenon lamp	leica	DMI6000
camera	hamamatsu	8100601
metafluor/Mmfluor software		11640905
pH sensitive probe, BCECF-AM	life technologies	B1170
Nigericin	Sigma	N7143