Extracellular Cations Research Articles

Divalent Cations and Lipid Composition Modulate Membrane Insertion and Cancer-Targeting Action of pHLIP

The pH-Low Insertion Peptide (pHLIP) has emerged as an important tool for targeting cancer cells. It has been assumed that its targeting mechanism depends solely on the mild acidic environment surrounding tumors. Here, we examine the role of the two predominant extracellular divalent cations, Ca2+ and Mg2+, on pHLIP's cellular targeting, insertion, and drug delivery. Using a combination of cellular and spectroscopic techniques we demonstrate that physiologically-relevant concentrations of either cation drastically promote the interaction and insertion of pHLIP.

The Sodium Leak Channel Complex is Modulated by Voltage and Extracellular Calcium

Summary The sodium leak channel (NALCN) is essential for survival in mammals: NALCN mutations are life-threatening in humans and knockout is lethal in mice. However, the basic functional and pharmacological properties of NALCN have remained elusive. Here, we found that the robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80 and FAM155A. The resulting NALCN channel complex is constitutively active, conducts monovalent cations but is blocked by physiological concentrations of extracellular divalent cations. Our data support the notion that NALCN is directly responsible for the increased excitability observed in a variety of neurons in reduced extracellular Ca2+. Despite the smaller number of voltage-sensing residues in the putative voltage sensors of NALCN, the channel complex shows voltage-dependent modulation of the constitutive current, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points towards formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting. Highlights Function of NALCN requires UNC79, UNC80 and FAM155A The complex is permeable to monovalent cations, but is blocked by divalent cations The complex displays a constitutively active, voltage-modulated current phenotype Positively charged side chains in S4 of NALCN VSD I and II confer voltage sensitivity

The transcriptional correlates of divergent electric organ discharges in Paramormyrops electric fish

BackgroundUnderstanding the genomic basis of phenotypic diversity can be greatly facilitated by examining adaptive radiations with hypervariable traits. In this study, we focus on a rapidly diverged species group of mormyrid electric fish in the genus Paramormyrops, which are characterized by extensive phenotypic variation in electric organ discharges (EODs). The main components of EOD diversity are waveform duration, complexity and polarity. Using an RNA-sequencing based approach, we sought to identify gene expression correlates for each of these EOD waveform features by comparing 11 specimens of Paramormyrops that exhibit variation in these features.ResultsPatterns of gene expression among Paramormyrops are highly correlated, and 3274 genes (16%) were differentially expressed. Using our most restrictive criteria, we detected 145–183 differentially expressed genes correlated with each EOD feature, with little overlap between them. The predicted functions of several of these genes are related to extracellular matrix, cation homeostasis, lipid metabolism, and cytoskeletal and sarcomeric proteins. These genes are of significant interest given the known morphological differences between electric organs that underlie differences in the EOD waveform features studied.ConclusionsIn this study, we identified plausible candidate genes that may contribute to phenotypic differences in EOD waveforms among a rapidly diverged group of mormyrid electric fish. These genes may be important targets of selection in the evolution of species-specific differences in mate-recognition signals.

Effect of plant flavonoids on the volume regulation of rat thymocytes under hypoosmotic stress.

Cell volume regulation and volume-regulated anion channels are critical for cell survival in non-isosmotic conditions, and dysregulation of this system is detrimental. Although genes and proteins underlying this basic cellular machinery were recently identified, the pharmacology remains poorly explored. We examined effects of 16 flavonoids on the regulatory volume decrease (RVD) of thymocytes under hypoosmotic stress assessed by light transmittance and on the activity of volume-sensitive chloride channel by patch-clamp technique. Comparison of effects of flavonoids on RVD revealed a group of four active substances with lehmannin being the strongest inhibitor (IC50 = 8.8 μM). Structure-functional comparison suggested that hydrophobicity brought about by methoxy, prenyl or lavandulyl groups as well as by the absence of glucosyl fragment together with localization of the phenyl ring B at the position C2 (which is at C3 in totally inactive isoflavones) are important structural determinants for the flavonoids activity as volume regulation inhibitors. All active flavonoids suppressed RVD under Gramicidin D-NMDG hypotonic stress conditions when cationic permeability was increased by an ionophore, gramicidin D, with all extracellular monovalent cations replaced with bulky NMDG+ suggesting that they target volume-sensitive anionic permeability. While effects of hispidulin and pulicarin were only partial, lehmannin and pinocembrin completely abolished RVD under Gramicidin D-NMDG conditions. In direct patch-clamp experiments, lehmannin and pinocembrin produced a strong inhibiting effect on the swelling-induced whole-cell chloride conductance in a voltage-independent manner. Lehmannin, pinocembrin, and possibly hispidulin and pulicarin may serve as leads for developing effective low-toxic immunomodulators.

Altered allostery of the left flipper domain underlies the weak ATP response of rat P2X5 receptors

Although the extracellular ATP-gated cation channel purinergic receptor P2X5 is widely expressed in heart, skeletal muscle, and immune and nervous systems in mammals, little is known about its functions and channel-gating activities. This lack of knowledge is due to P2X5's weak ATP responses in several mammalian species, such as humans, rats, and mice. WT human P2X5 (hP2X5Δ328-349) does not respond to ATP, whereas a full-length variant, hP2X5 (hP2X5-FL), containing exon 10 encoding the second hP2X5 transmembrane domain (TM2), does. However, although rat P2X5 (rP2X5) has a full-length TM2, ATP induces only weak currents in rP2X5, which prompted us to investigate the mechanism underlying this small ATP response. Here, we show that single replacements of specific rP2X5 residues with the corresponding residues in hP2X5 (S191F or F195H) significantly enhance the current amplitude of rP2X5. Using a combination of engineered disulfide cross-linking, single-channel recording, and molecular modeling, we interrogated the effects of S191F and F195H substitutions on the allostery of the left flipper (LF) domain. On the basis of our findings, we propose that the bound ATP-induced distinct allostery of the LF domain with that of other functional subtypes has caused the weak ATP response of rP2X5 receptors. The findings of our study provide the prerequisite for future transgenic studies on the physiological and pathological functions of P2X5 receptors.

Structural determinants underlying permeant discrimination of the Cx43 hemichannel

Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.

Functional expression of TRPM7 as a Ca2+ influx pathway in adipocytes.

In adipocytes, intracellular Ca2+ and Mg2+ modulates physiological functions, such as insulin action and the secretion of adipokines. TRPM7 is a Ca2+/Mg2+‐permeable non‐selective cation channel. TRPM7 mRNA is highly expressed in adipose tissue, however, its functional expression in adipocytes remains to be elucidated. In this study, we demonstrated for the first time that TRPM7 was functionally expressed in both freshly isolated white adipocytes and in 3T3‐L1 adipocytes differentiated from a 3T3‐L1 pre‐adipocyte cell line by whole‐cell patch‐clamp recordings. Consistent with known properties of TRPM7 current, the current in adipocytes was activated by the elimination of extracellular divalent cations and the reduction of intracellular free Mg2+ concentrations, and was inhibited by the TRPM7 inhibitors, 2‐aminoethyl diphenylborinate (2‐APB), hydrogen peroxide (H2O2), N‐methyl maleimide (NMM), NS8593, and 2‐amino‐2‐[2‐(4‐octylphenyl)ethyl]‐1,3‐propanediol (FTY720). Treatment with small‐interfering (si) RNA targeting TRPM7 resulted in a reduction in the current to 23 ± 7% of nontargeting siRNA‐treated adipocytes. Moreover a TRPM7 activator, naltriben, increased the TRPM7‐like current and [Ca2+]i in 3T3‐L1 adipocytes but not in TRPM7‐knockdown adipocytes. These findings indicate that TRPM7 is functionally expressed, and plays a role as a Ca2+ influx pathway in adipocytes.

An extracellular cation coordination site influences ion conduction of OsHKT2;2

BackgroundHKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species.ResultsBy analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results.ConclusionWe identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.

Physicochemical mechanotransduction alters nuclear shape and mechanics via heterochromatin formation.

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels (MSCs), without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through MSCs and consequent changes to histone modification state and chromatin-based nuclear rigidity.

Physicochemical mechanotransduction alters nuclear shape and mechanics via heterochromatin formation.

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels, without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through mechanosensitive ion channels and consequent changes to histone modification state and chromatin-based nuclear rigidity.

APH-1A Component of γ-Secretase Forms an Internal Water and Ion-Containing Cavity.

Anterior pharynx-defective 1A (APH-1A) is a seven transmembrane component of γ-secretase (GS), an aspartyl protease enzyme involved in the production of toxic amyloid-β peptides in Alzheimer's disease patients. Cryo-electron microscopy structures of the enzyme complex revealed a central cavity in its APH-1A component, similar to water-containing cavities in G-protein coupled receptors (GPCRs). In this work, we performed molecular dynamics and umbrella sampling simulations to understand the role of the APH-1A cavity in the GS complex. Our results suggest that APH-1A is able to store water molecules in its inner cavity and transport some of them between cell spaces. Additionally, APH-1A allows the influx of extracellular cations into a central hydrophilic cavity but cannot transport them into the intracellular space. Overall, this study seeks to describe an alternative APH-1A function in GS besides its complex stabilization role and provide novel approaches to understand the functioning of the GS enzyme.

Removal of Microcystis blooms using enhanced colony formation and buoyancy by controlling extracellular polysaccharides and cation concentrations

This study attempted to promote the colony formation and enlarge the colony size of wild Microcystis strain by adjusting extracellular polysaccharides and cationic ion concentrations in culture medium, and then, using the enhanced floating velocity (Stokes’ law), wild Microcystis at the water surface was removed. In order to examine the difference between extracellular polysaccharides mixture and tightly cell-bound extracellular polysaccharides used in this study, the component composition and physical properties were also examined. The results showed that tightly cell-bound extracellular polysaccharides contained the greater amounts of cations (Ca2+ and Mg2+), phosphorus, carboxy group and monosaccharides than extracellular polysaccharides mixture. There were greater amounts of carboxy groups and mannose residue in tightly cell-bound extracellular polysaccharides than extracellular polysaccharides mixture, which would contribute to the stronger cell–cell adhesion of wild Microcystis to make colony formation and expand colony size. The adjustment of extracellular polysaccharides and cationic ion concentrations was effective method to promote the colony formation and enlarge the colony size of wild Microcystis, and that the control of Microcystis buoyancy by enhancing the colony size would be one of the options for the suppression of Microcystis blooms from the viewpoint of cost-effective, low-energy, environmentally-friendly, and non-destructive of Microcystis cells.

Turning complexity into clarity: polyuria and hypernatremia

Turning complexity into clarity: polyuria and hypernatremia

Molecular mechanism underlying the subtype-selectivity of competitive inhibitor NF110 and its distinct potencies in human and rat P2X3 receptors

P2X receptors are a family of extracellular ATP-gated trimeric cation channels that is widely distributed in human tissues. Quite some drug candidates targeting P2X receptors have entered into preclinical or main phases of clinical trials, but many of them failed due to low subtype-selectivity or species differences in pharmacological activities between human and experimental animals. Here, we identified the distinct inhibitory efficacies of NF110, a competitive inhibitor, between the rat (rP2X3) and human (hP2X3) P2X3 receptors. We demonstrated that this difference is determined by two amino acids located in the dorsal fin (DF) domain of P2X3 receptors. As revealed by mutagenesis, metadynamics, and covalent modification, NF110-mediated rP2X3 inhibition may be through a filling in the cavity formed by the DF, left flipper (LF) and lower body (LB) to partially, rather than fully, occupy the ATP-binding pocket. Moreover, substitution of residues located in the DF and/or LF domains of the rP2X2 receptor, a NF110-insensitive subtype, with the equivalent amino acids of rP2X3, bestowed the sensitivity of rP2X2 to NF110. The critical roles of the DF and LF domains in channel gating of P2X and low conservativity in residue sequences of those two domains raise the possibility that small molecules differentially interacting with the residues of the DF and LF domains of different P2X receptors may modulate channel’s activity in a subtype-selective manner. However, the possible species-specificity of P2X inhibitors/modulators makes it more complex when interpreting the preclinical data into clinical researches. Nevertheless, our data provide new insights into the subtype-selectivity of competitive inhibitors and their distinct potencies in the human and experimental animals, both of which are extremely important in the drug discovery of P2X receptors.

Water bicarbonate modulates the response of the shore crab Carcinus maenas to ocean acidification.

Ocean acidification causes an accumulation of CO2 in marine organisms and leads to shifts in acid-base parameters. Acid-base regulation in gill breathers involves a net increase of internal bicarbonate levels through transmembrane ion exchange with the surrounding water. Successful maintenance of body fluid pH depends on the functional capacity of ion-exchange mechanisms and associated energy budget. For a detailed understanding of the dependence of acid-base regulation on water parameters, we investigated the physiological responses of the shore crab Carcinus maenas to 4weeks of ocean acidification [OA, P(CO2)w = 1800µatm], at variable water bicarbonate levels, paralleled by changes in water pH. Cardiovascular performance was determined together with extra-(pHe) and intracellular pH (pHi), oxygen consumption, haemolymph CO2 parameters, and ion composition. High water P(CO2) caused haemolymph P(CO2) to rise, but pHe and pHi remained constant due to increased haemolymph and cellular [HCO3-]. This process was effective even under reduced seawater pH and bicarbonate concentrations. While extracellular cation concentrations increased throughout, anion levels remained constant or decreased. Despite similar levels of haemolymph pH and ion concentrations under OA, metabolic rates, and haemolymph flow were significantly depressed by 40 and 30%, respectively, when OA was combined with reduced seawater [HCO3-] and pH. Our findings suggest an influence of water bicarbonate levels on metabolic rates as well as on correlations between blood flow and pHe. This previously unknown phenomenon should direct attention to pathways of acid-base regulation and their potential feedback on whole-animal energy demand, in relation with changing seawater carbonate parameters.

Subtype-selective inhibition of acid-sensing ion channel 3 by a natural flavonoid.

Acid-sensing ion channels (ASICs) are extracellular proton-gated cation channels that have been implicated in multiple physiological and pathological processes, and peripheral ASIC3 prominently participate into the pathogenesis of chronic pain, itch, and neuroinflammation, which necessitates the need for discovery and development of novel modulators in a subtype-specific manner. Whole-cell patch clamp recordings and behavioral assays were used to examine the effect of several natural compounds on the ASIC-mediated currents and acid-induced nocifensive behavior, respectively. We identified a natural flavonoid compound, (-)-epigallocatechin gallate (EGCG, compound 11), that acts as a potent inhibitor for the ASIC3 channel in an isoform-specific way. The compound 11 inhibited ASIC3 currents with an apparent half maximal inhibitory concentration of 13.2μmol/L when measured at pH 5.0. However, at the concentration up to 100μmol/L, the compound 11 had no significant impacts on the homomeric ASIC1a, 1b, and 2a channels. In contrast to most of the known ASIC inhibitors that usually bear either basic or carboxylic groups, the compound 11 belongs to the polyphenolic family. In compound 11, both the chirality and the 3-hydroxyl group of its pyrogallol part, in addition to the integrity of the gallate part, are crucial for the inhibitory efficacy. Finally, EGCG was found significantly to decrease the acid-induced nocifensive behavior in mice. Taken together, these results thus defined a novel backbone structure for small molecule drug design targeting ASIC3 channels to treat pain-related diseases.

EDL Gated FET Biosensor Array for the Investigation of Ion Channels and Bioelectric Signals of Circulating Tumor Cells

This paper demonstrates the applications of novel high sensitive electrical double layer (EDL) gated FET biosensor array in detecting and counting cells, and analyzing their bioelectric signals in different test conditions. We focused on extremely rare cell type such as Circulating Tumor Cells (CTCs) (HCT-8 cell line) derived from Colorectal Cancer (CRC) cell line. CRC is one of the major forms of cancer that affects human population and CTCs have emerged as a clinical biomarker for diagnosis and prognosis of metastatic CRC. Previously, we had shown that our methodology can detect and count CTCs with very high sensitivity, with up to single cell resolution. Simultaneously, we discovered our sensor platform can be used to dynamically monitor the changes in transmembrane potential (Vm) of the cell, which is an important fundamental bioelectric signal of cell. Studies have shown Vm may be useful as a primary parameter in understanding disease progression, cell signaling mechanism and ion channel gating. We demonstrated that the EDL gated FET sensor can dynamically monitor the changes in Vm induced by high concentrations of extracellular KCl. It was also shown that the sensor response changes as extracellular divalent cation concentrations are dynamically increased towards physiological concentrations. The results display the ability of the sensing platform to monitor the cellular response to dynamically changing, very low ion concentrations as well. Furthermore, an interesting application of bioelectric signal study is demonstrated using cadmium ions to block the calcium channels present in the cell. As shown in figure, in low micro molar concentrations, cadmium acts as a permeant channel blocker (electrically, the cell is depolarized and as the Cd2+ concentration increases, it re-polarizes) and in higher concentrations, it accumulates in the intracellular region (the cell is depolarized again), causing induction of apoptotic signals. The sensor response closely follows the cellular response to permeant channel blockers such as cadmium. These results demonstrate that our sensing methodology can replace the conventional electrophysiological methods or optical potentiometric probes to study fundamental cellular mechanisms such as ion channel gating. Our method offers non-invasive means to monitor changes in Vm, with very high sensitivity for individual cells. The cell based studies can be carried out directly in physiological media with high ionic strength, without the use of extensive pre-treatments or additional reagents. Figure 1

EDL Gated FET Biosensor Array for the Investigation of Ion Channels and Bioelectric Signals of Circulating Tumor Cells

This paper demonstrates a novel methodology to analyze the bioelectric signals of cells in different test media using highly sensitive Electrical Double Layer (EDL) gated AlGaN/GaN HEMT (High Electron Mobility Transistor) biosensor array. Circulating Tumor Cells (CTCs) derived from Colorectal Cancer (CRC) were the target of this experiment. Previously, we had demonstrated that our sensor can detect and count the number of captured cells with up to single cell resolution. Thereafter, we further discovered that our sensor can be used in detecting important fundamental bioelectric signals of cell. We have demonstrated that our FET (Field Effect Transistor) biosensor can dynamically monitor the changes in transmembrane potential of cells induced by high concentrations of extracellular KCl, low and high extracellular divalent cation concentrations. Additionally, cadmium ions were used to block the calcium channels present in the cell demonstrating an interesting application of bioelectric signal study. Thus, we demonstrate that our sensing methodology can replace the conventional methods to study fundamental cellular mechanisms such as ion channel gating.

Membrane Lipid Compositions Control Dye-Permeable Pore of the P2X7 Receptor

The P2X7 receptor is an extracellular ATP-gated cation channel. Activation of P2X7 leads to the opening of a dye permeable pore, which causes cell lysis and eventually results in various health problems including chronic pain, neurodegeneration, and tumor growth. However, it remains controversial what constitutes this dye-permeable pore and how it opens. Two prevailing hypotheses are: 1) P2X7 dilates its transmembrane channel to form a larger pore and 2) P2X7 activation recruits other large conductance channels. To clarify whether P2X7 itself constitutes a dye permeable pore, we purified and reconstituted P2X7 into liposomes and measured pore activity. We observed strong dye uptake from P2X7 reconstituted liposomes, suggesting that P2X7 can form a dye permeable pore in the absence of other cellular components. To our surprise, dye uptake was independent of the C-terminal domain, which has been considered to be absolutely necessary for pore formation. Furthermore, dye uptake was immediately observed after ATP application, indicating that pore dilation is unlikely. We also discovered that the P2X7-dependent pore was facilitated by phosphatidylglycerol and sphingomyelin but strongly inhibited by cholesterol through direct interactions with the transmembrane domain of P2X7. In combination with a fatty acid incorporation assay, our results suggest that palmitoylation at the C-terminal domain counteracts the inhibitory effect of cholesterol by facilitating P2X7 channel opening.

Cpx-dependent expression of YqjA requires cations at elevated pH.

Under alkaline pH conditions, Escherichia coli must maintain a stable cytoplasmic pH of about 7.6 that is acidic relative to the environment. Bacteria employ various mechanisms to survive alkaline pH; however, membrane cation/H+ antiporters play a primary role by facilitating inward transport of protons. Escherichia coli YqjA belongs to the DedA/Tvp38 membrane protein family and, along with its paralog YghB, is required for growth at 42°C, proper cell division and antibiotic resistance. YqjA is required for viability at alkaline pH, requiring cations sodium or potassium to support growth under these conditions, suggesting it may be a transporter. We measured yqjA expression at different pHs and cation concentrations using a yqjA promoter-lacZ fusion. We found that yqjA promoter activity was highest at alkaline pH. Increased activity of the yqjA promoter required both the transcriptional regulator CpxR, in agreement with previous results, and sodium or potassium salts at alkaline pH. Extracellular cations are also required for activity of cpxP and degP promoters at alkaline pH, suggesting this is a general property of the Cpx regulon. To our knowledge, this is the first demonstration of cation-dependent expression of Cpx-regulated genes at alkaline pH.