REVIEWS Sensors and regulators of intracellular pH Joseph R. Casey*, Sergio Grinstein‡ and John Orlowski§ Abstract | Protons dictate the charge and structure of macromolecules and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce or consume proton equivalents. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well. Proton-motive force Eukaryotic cells are highly compartmentalized, seg- As a result, βintrinsic is comparatively low at physiological regating specific functions within membrane-bound pH (10–20 mM at pH 7.2), increasing at more extreme (ψH+). The driving force for proton (or equivalent) organelles. Compartmentalization probably evolved from values (for example, 40 mM at pH 6.4)3. This apparent movement, consisting of the the need to provide distinct environmental conditions shortcoming is alleviated by the second component of the proton concentration gradient for the optimal operation of individual metabolic pathways, total buffering capacity, β . Mammalian cells are con- and the transmembrane HCO3– electrical potential. and also to store energy as electrochemical gradients tinuously exposed to CO2, which is uncharged and readily across the membrane dielectric. Protons (and proton traverses most biological membranes4. The hydration of pH buffering capacity equivalents) have a crucial role in this context. Virtually CO2 and the subsequent deprotonation of carbonic acid A measure of the ability of a all proteins depend on pH to maintain their structure generate HCO –, an effective proton buffer (BOX 1). At the solution to withstand changes 3 in pH. It is defined as and function, and protonation–deprotonation events dic- prevailing CO2 pressure (PCO2; ~37 mm Hg ) and using BOX 1 β= dn/dpH, where n is the tate the charge of biological surfaces and are an integral the equation in , βHCO3– can be calculated to contribute number of acid or base part of many metabolic reactions1. Moreover, the proton- 29 mM to the total buffering capacity at pH 7.1. equivalents that need to be motive force (ψH+) is key to the generation and conver- Together, βintrinsic and βHCO3– reduce the impact of acute added to alter pH. sion of cellular energy. It is therefore not surprising that challenges on the intracellular pH, but are insufficient to intracellular pH is stringently regulated, nor that it varies counteract sustained stress. In the absence of other regu- greatly among the different organelles (FIG. 1). The objec- latory processes, the continuous generation of metabolic tive of this Review is to provide an up-to-date overview acid equivalents, together with the ongoing transport of + – – of the sensors, determinants and regulators of the pH of ions that alter the pH (for example, H , OH and HCO3 ), individual subcellular compartments. For simplicity, our would promptly consume the passive (physicochemical) discussion will concentrate on the mechanisms that are buffers, which are finite. Therefore other, more dynamic present in most non-polarized mammalian cells, address- and sustained mechanisms are required to ensure long- ing only occasionally the unique, but no less important, term pH homeostasis. As discussed in detail below, vari- systems that have been developed by epithelial and other ous different, often redundant, mechanisms have evolved *Departments of Physiology and Biochemistry, highly specialized cells. to regulate pH in individual compartments. University of Alberta, Canada. Buffering power: rapid but finite Cytosolic pH ‡Cell Biology Program, Because of its paramount importance, the pH of the cell Under physiological conditions, the extracellular pH is The Hospital for Sick Children, Toronto, Canada. is defended at multiple levels. Cellular compartments are slightly alkaline (~7.3–7.4). However, even when bathed §Department of Physiology, protected from rapid, localized pH swings by their inher- in large volumes of this heavily buffered, alkaline medium, pH buffering capacity (FIG. 1) McGill University, Montreal, ent (β). The total buffer capacity (βtotal) the cytosolic pH (pHc) is slightly more acidic and, Canada. consists of two components: βintrinsic and βHCO3–. βintrinsic is in fact, the cells have to guard against further acidification. Correspondence to S.G. provided by various intracellular weak acids and bases, Two reasons account for the tendency of the cytosol to e-mail: [email protected] doi:10.1038/nrm2820 including phosphate groups and side chains of amino acidify. First, the electrical potential across the membrane, 2 pK Published online acids . Remarkably, the a values of most ionizable groups which is negative inside, drives the uptake of positively 9 December 2009 in the cell are considerably above or below neutrality. charged protons and the efflux of negatively charged 50 | JANUARY 2010 | VOLUME 11 www.nature.com/reviews/molcellbio REVIEWS membrane of some epithelial cells and in osteoclasts. These cells express on their surface vacuolar-type + Peroxisomes Secretory (V) electrogenic H -ATP hydrolases (V-ATPases) — 7 granule 5.5 energy-driven active proton pumps that are similar to Mitochondria Recycling Early those found in endomembranes (see below) and are 8 endosome endosome 6.5 6.3 characteristically sensitive to macrolide antibiotics such Trans-golgi 6 network 6 as bafilomycin . The acid-secreting parietal cells of the Trans stomach and distal tubule cells of the kidney addition- Golgi Late ally express a different type of ATPase, which exchanges network Medial endosome K+ for H+ in an electroneutral manner7. Although these 5.5 Cis ATPases effectively extrude protons from the cytosol, 6.7 Lysosome their primary role is not the regulation of intracellular Endoplasmic 4.7 reticulum 7.2 pH. Osteoclasts secrete acid into lacunae, where bone is resorbed, whereas the gastric and distal tubular Nucleus Cytosol ATPases, which are restricted to the apical surface of 7.2 7.2 the cells, are intended for trans epithelial delivery of pro- tons to the lumen of the stomach and the renal tubules, Figure 1 | pH of the different subcellular compartments. The pH of individual respectively7,8. Thus, proton-pumping ATPases are not cellular organelles and compartments in a prototypicalNatur mammaliane Reviews | cell.Molecular The values Cell Biolog werey important participants in the regulation of pHc in most collected from various sources. The mitochondrial pH refers to the matrix, that is, the mammalian cells. Instead, protons are extruded from the space contained by the inner mitochondrial membrane. Early endosomes refer to the cells against their electrochemical gradient by coupling sorting endosomal compartment. The pH of the multivesicular late endosome refers to to other substrates through exchangers (antiporters) or the bulk luminal fluid; the pH of the fluid contained by the internal vesicles might differ. co-transporters (symporters) (FIG. 2). – + + bases, such as HCO3 , through conductive pathways. Alkali cation–H exchangers. Alkali cation–H exchangers Second, net acid equivalents are generated by various (FIG. 2) are homodimeric complexes that directly couple the metabolic reactions (for example, ATP production in the transfer of H+ across biological membranes to the counter- cytoplasm by glycolysis and in mitochondria by oxidative transport of monovalent cations such as Na+ or K+, and phosphorylation; see FIG. 2), a situation that is exacerbated are an evolutionarily conserved mechanism for protect- during bursts of activity, such as in muscle contraction or ing cells against excess acidification. Mammals possess on activation of leukocytes by pathogens5. at least eleven distinct orthologues: Na+–H+ exchanger 1 To prevent their gradual accumulation, protons (NHE1; also known as SLC9A1)–NHE9 (also known must be continuously and actively extruded from the as SLC9A9), Na+–H+ antiporter 1 (NHA1; also known as cytosol across the plasma membrane. The energy for NHEDC1) and NHA2 (also known as NHEDC2). proton extrusion can be provided directly or indirectly These are expressed in a ubiquitous or tissue-specific by ATP. Proton-pumping ATPases exist in the plasma manner (see Supplementary information S1 (table))9,10. – Box 1 | CO2, HCO3 and the regulation of pH – + Eukaryotic cells constantly produce CO2, an end product of HCO3 + H CO2 + H2O mitochondrial energy production. In the presence of water, CO2 is effectively a conjugate acid by virtue of the following reactions: CO + H O ↔ H CO ↔ HCO – + H+ 2 2 2 3 3 CA12 – Inter-conversion of CO2 and HCO3 occurs spontaneously. Carbonic anhydrase (CA) enzymes (see the figure), however, greatly accelerate the reaction, with catalytic CO2 rates of up to 106 s–1 (REF.117). The human genome encodes 11 different carbonic anhydrases117. Since they catalyse the Plasma membrane production or consumption of H+ (depending on the prevailing substrate concentrations), carbonic anhydrase isoforms exercise some degree of control on the kinetics CA2 and location of pH changes. Their function is likely to differ depending on their site of expression: some isoforms are pKa The acid dissociation constant. located in the cytosol (CA1, CA2, CA3, CA7 and CA8), Cytosol CO2 + H2O A quantitative