Proton Transport in Cancer Cells: The Role of Carbonic Anhydrases.

Intra- and extracellular pH regulation is a pivotal function of all cells and tissues. Net outward transport of H+ is a prerequisite for normal physiological function, since a number of intracellular processes, such as metabolism and energy supply, produce acid. In tumor tissues, distorted pH regulation results in extracellular acidification and the formation of a hostile environment in which cancer cells can outcompete healthy local host cells. Cancer cells employ a variety of H+/HCO3--coupled transporters in combination with intra- and extracellular carbonic anhydrase (CA) isoforms, to alter intra- and extracellular pH to values that promote tumor progression. Many of the transporters could closely associate to CAs, to form a protein complex coined "transport metabolon". While transport metabolons built with HCO3--coupled transporters require CA catalytic activity, transport metabolons with monocarboxylate transporters (MCTs) operate independently from CA catalytic function. In this article, we assess some of the processes and functions of CAs for tumor pH regulation and discuss the role of intra- and extracellular pH regulation for cancer pathogenesis and therapeutic intervention.

14-3-3 Proteins and Other Candidates form Protein-Protein Interactions with the Cytosolic C-terminal End of SOS1 Affecting Its Transport Activity.

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na+ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program.

Transport Metabolons and Acid/Base Balance in Tumor Cells.

Solid tumors are metabolically highly active tissues, which produce large amounts of acid. The acid/base balance in tumor cells is regulated by the concerted interplay between a variety of membrane transporters and carbonic anhydrases (CAs), which cooperate to produce an alkaline intracellular, and an acidic extracellular, environment, in which cancer cells can outcompete their adjacent host cells. Many acid/base transporters form a structural and functional complex with CAs, coined "transport metabolon". Transport metabolons with bicarbonate transporters require the binding of CA to the transporter and CA enzymatic activity. In cancer cells, these bicarbonate transport metabolons have been attributed a role in pH regulation and cell migration. Another type of transport metabolon is formed between CAs and monocarboxylate transporters, which mediate proton-coupled lactate transport across the cell membrane. In this complex, CAs function as "proton antenna" for the transporter, which mediate the rapid exchange of protons between the transporter and the surroundings. These transport metabolons do not require CA catalytic activity, and support the rapid efflux of lactate and protons from hypoxic cancer cells to allow sustained glycolytic activity and cell proliferation. Due to their prominent role in tumor acid/base regulation and metabolism, transport metabolons might be promising drug targets for new approaches in cancer therapy.

Energy Dynamics in the Brain: Contributions of Astrocytes to Metabolism and pH Homeostasis.

Regulation of metabolism is complex and involves enzymes and membrane transporters, which form networks to support energy dynamics. Lactate, as a metabolic intermediate from glucose or glycogen breakdown, appears to play a major role as additional energetic substrate, which is shuttled between glycolytic and oxidative cells, both under hypoxic and normoxic conditions. Transport of lactate across the cell membrane is mediated by monocarboxylate transporters (MCTs) in cotransport with H+, which is a substrate, a signal and a modulator of metabolic processes. MCTs form a "transport metabolon" with carbonic anhydrases (CAs), which not only provide a rapid equilibrium between CO2, HCO3 - and H+, but, in addition, enhances lactate transport, as found in Xenopus oocytes, employed as heterologous expression system, as well as in astrocytes and cancer cells. Functional interactions between different CA isoforms and MCTs have been found to be isoform-specific, independent of the enzyme's catalytic activity, and they require physical interaction between the proteins. CAs mediate between different states of metabolic acidosis, induced by glycolysis and oxidative phosphorylation, and play a relay function in coupling pH regulation and metabolism. In the brain, metabolic processes in astrocytes appear to be linked to bicarbonate transport and to neuronal activity. Here, we focus on physiological processes of energy dynamics in astrocytes as well as on the transfer of energetic substrates to neurons.

Functional expression of electrogenic sodium bicarbonate cotransporter 1 (NBCe1) in mouse cortical astrocytes is dependent on S255-257 and regulated by mTOR.

The electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), is the major bicarbonate transporter expressed in astrocytes. It is highly sensitive for bicarbonate and the main regulator of intracellular, extracellular, and synaptic pH, thereby modulating neuronal excitability. However, despite these essential functions, the molecular mechanisms underlying NBCe1-mediated astrocytic response to extracellular pH changes are mostly unknown. Using primary mouse cortical astrocyte cultures, we investigated the effect of long-term extracellular metabolic alkalosis on regulation of NBCe1 and elucidated the underlying molecular mechanisms by immunoblotting, biotinylation of surface proteins, intracellular H+ recording using the H+ -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, and phosphoproteomic analysis. The results showed significant downregulation of NBCe1 activity following metabolic alkalosis without influencing protein abundance or surface expression of NBCe1. During alkalosis, the rate of intracellular H+ changes upon challenging NBCe1 was decreased in wild-type astrocytes, but not in cortical astrocytes from NBCe1-deficient mice. Alkalosis-induced decrease of NBCe1 activity was rescued after activation of mTOR signaling. Moreover, mass spectrometry revealed constitutively phosphorylated S255-257 and mutational analysis uncovered these residues being crucial for NBCe1 transport activity. Our results demonstrate a novel mTOR-regulated mechanism by which NBCe1 functional expression is regulated. Such mechanism likely applies not only for NBCe1 in astrocytes, but in epithelial cells as well.

Catalytically inactive carbonic anhydrase-related proteins enhance transport of lactate by MCT1.

Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to protons and bicarbonate and thereby play a fundamental role in the epithelial acid/base transport mechanisms serving fluid secretion and absorption for whole-body acid/base regulation. The three carbonic anhydrase-related proteins (CARPs) VIII, X, and XI, however, are catalytically inactive. Previous work has shown that some CA isoforms noncatalytically enhance lactate transport through various monocarboxylate transporters (MCT). Therefore, we examined whether the catalytically inactive CARPs play a role in lactate transport. Here, we report that CARP VIII, X, and XI enhance transport activity of the MCT MCT1 when coexpressed in Xenopus oocytes, as evidenced by the rate of rise in intracellular H+ concentration detected using ion-sensitive microelectrodes. Based on previous studies, we suggest that CARPs may function as a 'proton antenna' for MCT1, to drive proton-coupled lactate transport across the cell membrane.

Tight coupling of astrocyte energy metabolism to synaptic activity revealed by genetically encoded FRET nanosensors in hippocampal tissue.

The potassium ion, K+, a neuronal signal that is released during excitatory synaptic activity, produces acute activation of glucose consumption in cultured astrocytes, a phenomenon mediated by the sodium bicarbonate cotransporter NBCe1 ( SLC4A4). We have explored here the relevance of this mechanism in brain tissue by imaging the effect of neuronal activity on pH, glucose, pyruvate and lactate dynamics in hippocampal astrocytes using BCECF and FRET nanosensors. Electrical stimulation of Schaffer collaterals produced fast activation of glucose consumption in astrocytes with a parallel increase in intracellular pyruvate and biphasic changes in lactate . These responses were blocked by TTX and were absent in tissue slices prepared from NBCe1-KO mice. Direct depolarization of astrocytes with elevated extracellular K+ or Ba2+ mimicked the metabolic effects of electrical stimulation. We conclude that the glycolytic pathway of astrocytes in situ is acutely sensitive to neuronal activity, and that extracellular K+ and the NBCe1 cotransporter are involved in metabolic crosstalk between neurons and astrocytes. Glycolytic activation of astrocytes in response to neuronal K+ helps to provide an adequate supply of lactate, a metabolite that is released by astrocytes and which acts as neuronal fuel and an intercellular signal.

Membrane-anchored carbonic anhydrase IV interacts with monocarboxylate transporters via their chaperones CD147 and GP70.

Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.

Cytosolic sodium regulation in mouse cortical astrocytes and its dependence on potassium and bicarbonate.

Sodium plays a major role in different astrocytic functions, including maintenance of ion homeostasis and uptake of neurotransmitters and metabolites, which are mediated by different Na+ -coupled transporters. In the current study, the role of an electrogenic sodium-bicarbonate cotransporter (NBCe1), a sodium-potassium-chloride transporter 1 (NKCC1) and sodium-potassium ATPase (Na+ -K+ -ATPase) for the maintenance of [Na+ ]i was investigated in cultured astrocytes of wild-type (WT) and of NBCe1-deficient (NBCe1-KO) mice using the Na+ -sensitive dye, asante sodium green-2. Our results suggest that cytosolic Na+ was higher in the presence of CO2 /HCO3- (15 mM) than CO2 /HCO3- -free, HEPES-buffered solution in WT, but not in NBCe1-KO astrocytes (12 mM). Surprisingly, there was a strong dependence of cytosolic [Na+ ] on the extracellular [HCO3- ] attributable to NBCe1 activity. Pharmacological blockage of NKCC1 with bumetanide led to a robust drop in cytosolic Na+ in both WT and NBCe1-KO astrocytes by up to 6 mM. There was a strong dependence of the cytosolic [Na+ ] on the extracellular [K+ ]. Inhibition of the Na+ -K+ -ATPase led to larger increase in cytosolic Na+ , both in the absence of K+ as compared with the presence of ouabain and in NBCe1-KO astrocytes as compared with WT astrocytes. Our results show that cytosolic Na+ in mouse cortical astrocytes can vary considerably and depends greatly on the concentrations of HCO3- and K+ , attributable to the activity of the Na+ -K+ -ATPase, of NBCe1 and NKCC1.

A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells.

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme's intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.

Neuronal control of astrocytic respiration through a variant of the Crabtree effect.

Aerobic glycolysis is a phenomenon that in the long term contributes to synaptic formation and growth, is reduced by normal aging, and correlates with amyloid beta deposition. Aerobic glycolysis starts within seconds of neural activity and it is not obvious why energetic efficiency should be compromised precisely when energy demand is highest. Using genetically encoded FRET nanosensors and real-time oxygen measurements in culture and in hippocampal slices, we show here that astrocytes respond to physiological extracellular K+ with an acute rise in cytosolic ATP and a parallel inhibition of oxygen consumption, explained by glycolytic stimulation via the Na+-bicarbonate cotransporter NBCe1. This control of mitochondrial respiration via glycolysis modulation is reminiscent of a phenomenon previously described in proliferating cells, known as the Crabtree effect. Fast brain aerobic glycolysis may be interpreted as a strategy whereby neurons manipulate neighboring astrocytes to obtain oxygen, thus maximizing information processing.

Modeling of pH regulation in tumor cells: Direct interaction between proton-coupled lactate transporters and cancer-associated carbonic anhydrase.

The most aggressive tumor cells, which often reside in a hypoxic environment, can release vast amounts of lactate and protons via monocarboxylate transporters (MCTs). This additional proton efflux exacerbates extracellular acidification and supports the formation of a hostile environment. In the present study we propose a novel, data-based model for this proton-coupled lactate transport in cancer cells. The mathematical settings involve systems coupling nonlinear ordinary and stochastic differential equations describing the dynamics of intra- and extracellular proton and lactate concentrations. The data involve time series of intracellular proton concentrations of normoxic and hypoxic MCF-7 breast cancer cells. The good agreement of our final model with the data suggests the existence of proton pools near the cell membrane, which can be controlled by intracellular and extracellular carbonic anhydrases to drive proton-coupled lactate transport across the plasma membrane of hypoxic cancer cells.

Reduction of epileptiform activity in ketogenic mice: The role of monocarboxylate transporters.

Epilepsy is a chronic neurological disorder that affects approximately 50 million people worldwide. Ketogenic diet (KD) can be a very effective treatment for intractable epilepsy. Potential mechanisms of action for KD have been proposed, including the re-balance among excitatory and inhibitory neurotransmission and decrease in the glycolytic rate in brain cells. KD has been shown to have an effect on the expression pattern of monocarboxylate transporters (MCT), however, it is unknown whether MCT transport activity is affected by KD and linked to the reduction of seizures during KD. Therefore, we studied the influence of KD on MCT transport activity and the role of MCTs during epileptiform activity. Our results showed a decrease in the epileptiform activity in cortical slices from mice fed on KD and in the presence of beta-hydroxybutyrate. KD increased transport capacity for ketone bodies and lactate in cortical astrocytes by raising the MCT1 expression level. Inhibition of MCT1 and MCT2 in control conditions decreases epileptiform activity, while in KD it induced an increase in epileptiform activity. Our results suggest that MCTs not only play an important role in the transport of ketone bodies, but also in the modulation of brain energy metabolism under normal and ketogenic conditions.

TGF-ß signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes.

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) expressed in astrocytes regulates intracellular and extracellular pH. Here, we introduce transforming growth factor beta (TGF-ß) as a novel regulator of NBCe1 transcription and functional expression. Using hippocampal slices and primary hippocampal and cortical astrocyte cultures, we investigated regulation of NBCe1 and elucidated the underlying signaling pathways by RT-PCR, immunoblotting, immunofluorescence, intracellular H(+ ) recording using the H(+ ) -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, mink lung epithelial cell (MLEC) assay, and chromatin immunoprecipitation. Activation of TGF-ß signaling significantly upregulated transcript, protein, and surface expression of NBCe1. These effects were TGF-ß receptor-mediated and suppressed following inhibition of JNK and Smad signaling. Moreover, 4-aminopyridine (4AP)-dependent NBCe1 regulation requires TGF-ß. TGF-ß increased the rate and amplitude of intracellular H+ changes upon challenging NBCe1 in wild-type astrocytes but not in cortical astrocytes from Slc4a4-deficient mice. A Smad4 binding sequence was identified in the NBCe1 promoter and Smad4 binding increased after activation of TGF-ß signaling. The data show for the first time that NBCe1 is a direct target of TGF-ß/Smad4 signaling. Through activation of the canonical pathway TGF-ß acts directly on NBCe1 by binding of Smad4 to the NBCe1 promoter and regulating its transcription, followed by increased protein expression and transport activity.

Bicarbonate sensing in mouse cortical astrocytes during extracellular acid/base disturbances.

KEY POINTS: The present study suggests that the electrogenic sodium-bicarbonate cotransporter, NBCe1, supported by carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes. This mechanism is proposed to play a major role in setting the pHi responses to extracellular acid/base challenges in astrocytes. A decrease in extracellular [HCO3- ] during isocapnic acidosis and isohydric hypocapnia, or an increase in intracellular [HCO3- ] during hypercapnic acidosis, was effectively sensed by NBCe1, which carried bicarbonate out of the cells under these conditions, and caused an acidification and sodium fall in WT astrocytes, but not in NBCe1-knockout astrocytes. Isocapnic acidosis, hypercapnic acidosis and isohydric hypocapnia evoked inward currents in NBCe1- and CAII-expressing Xenopus laevis oocytes, but not in native oocytes, suggesting that NBCe1 operates in the outwardly directed mode under these conditions consistent with our findings in astrocytes. We propose that bicarbonate sensing of astrocytes may have functional significance during extracellular acid/base disturbances in the brain, as it not only alters intracellular pH/[HCO3- ]-dependent functions of astrocytes, but also modulates the extracellular pH/[HCO3- ] in brain tissue. ABSTRACT: Extracellular acid/base status of the mammalian brain undergoes dynamic changes during many physiological and pathological events. Although intracellular pH (pHi ) of astrocytes responds to extracellular acid/base changes, the mechanisms mediating these changes have remained unresolved. We have previously shown that the electrogenic sodium-bicarbonate cotransporter, NBCe1, is a high-affinity bicarbonate carrier in cortical astrocytes. In the present study, we investigated whether NBCe1 plays a role in bicarbonate sensing in astrocytes, and in determining the pHi responses to extracellular acid/base challenges. We measured changes in intracellular H+ and Na+ in astrocytes from wild-type (WT) and from NBCe1-knockout (KO) mice, using ion-selective dyes, during isocapnic acidosis, hypercapnic acidosis and hypocapnia. We also analysed NBCe1-mediated membrane currents in Xenopus laevis oocytes under similar conditions. Comparing WT and NBCe1-KO astrocytes, we could dissect the contribution of NBCe1, of diffusion of CO2 across the cell membrane and, after blocking carbonic anhydrase (CA) activity with ethoxyzolamide, of the role of CA, for the amplitude and rate of acid/base fluxes. Our results suggest that NBCe1 transport activity in astrocytes, supported by CA activity, renders astrocytes bicarbonate sensors in the mouse cortex. NBCe1 carried bicarbonate into and out of the cell by sensing the variations of transmembrane [HCO3- ], irrespective of the changes in intra- and extracellular pH, and played a major role in setting pHi responses to the extracellular acid/base challenges. We propose that bicarbonate sensing of astrocytes may have potential functional significance during extracellular acid/base alterations in the brain.

Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail.

Mechanisms of CO2/H+ Sensitivity of Astrocytes.

Ventral regions of the medulla oblongata of the brainstem are populated by astrocytes sensitive to physiological changes in PCO2/[H+]. These astrocytes respond to decreases in pH with elevations in intracellular Ca2+ and facilitated exocytosis of ATP-containing vesicles. Released ATP propagates Ca2+ excitation among neighboring astrocytes and activates neurons of the brainstem respiratory network triggering adaptive increases in breathing. The mechanisms linking increases in extracellular and/or intracellular PCO2/[H+] with Ca2+ responses in chemosensitive astrocytes remain unknown. Fluorescent imaging of changes in [Na+]i and/or [Ca2+]i in individual astrocytes was performed in organotypic brainstem slice cultures and acute brainstem slices of adult rats. It was found that astroglial [Ca2+]i responses triggered by decreases in pH are preceded by Na+ entry, markedly reduced by inhibition of Na+/HCO3- cotransport (NBC) or Na+/Ca2+ exchange (NCX), and abolished in Na+-free medium or by combined NBC/NCX blockade. Acidification-induced [Ca2+]i responses were also dramatically reduced in brainstem astrocytes of mice deficient in the electrogenic Na+/HCO3- cotransporter NBCe1. Sensitivity of astrocytes to changes in pH was not affected by inhibition of Na+/H+ exchange or blockade of phospholipase C. These results suggest that in pH-sensitive astrocytes, acidification activates NBCe1, which brings Na+ inside the cell. Raising [Na+]i activates NCX to operate in a reverse mode, leading to Ca2+ entry followed by activation of downstream signaling pathways. Coupled NBC and NCX activities are, therefore, suggested to be responsible for functional CO2/H+ sensitivity of astrocytes that contribute to homeostatic regulation of brain parenchymal pH and control of breathing. SIGNIFICANCE STATEMENT: Brainstem astrocytes detect physiological changes in pH, activate neurons of the neighboring respiratory network, and contribute to the development of adaptive respiratory responses to the increases in the level of blood and brain PCO2/[H+]. The mechanisms underlying astroglial pH sensitivity remained unknown and here we show that in brainstem astrocytes acidification activates Na+/HCO3- cotransport, which brings Na+ inside the cell. Raising [Na+]i activates the Na+/Ca2+ exchanger to operate in a reverse mode leading to Ca2+ entry. This identifies a plausible mechanism of functional CO2/H+ sensitivity of brainstem astrocytes, which play an important role in homeostatic regulation of brain pH and control of breathing.

Selective inhibition of human carbonic anhydrase IX in Xenopus oocytes and MDA-MB-231 breast cancer cells.

Human carbonic anhydrase IX (CA IX) is overexpressed in the most aggressive and invasive tumors. Therefore, CA IX has become the promising antitumor drug target. Three inhibitors have been shown to selectively and with picomolar affinity inhibit human recombinant CA IX. Their inhibitory potencies were determined for the CA IX, CA II, CA IV and CA XII in Xenopus oocytes and MDA-MB-231 cancer cells. The inhibition IC50 value of microelectrode-monitored intracellular and extracellular acidification reached 15 nM for CA IX, but with no effect on CA II expressed in Xenopus oocytes. Results were confirmed by mass spectrometric gas analysis of lysed oocytes, when an inhibitory effect on CA IX catalytic activity was found after the injection of 1 nM VD11-4-2. Moreover, VD11-4-2 inhibited CA activity in MDA-MB-231 cancer cells at nanomolar concentrations. This combination of high selectivity and potency renders VD11-4-2, an auspicious therapeutic drug for target-specific tumor therapy.

Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies.

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na+ independent. Studies in oocytes and HEK293 cells could not detect Na+-coupled HCO3- transport or Cl-/HCO3- exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltage-dependent OH- or H+ movement was not measurable in SLC4A11-expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH-/H+ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H.

Aquaporin 4 as a NH3 Channel.

Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient <1 for NH4Cl at pH 8.0, at which pH an increased amount of the ammonia occurs in the form of NH3 Taken together with an NH4Cl-mediated intracellular alkalization (or lesser acidification) of AQP4-expressing oocytes, these data suggest that NH3 is able to permeate the pore of AQP4. Exposure to NH4Cl increased the membrane currents to a similar extent in uninjected oocytes and in oocytes expressing AQP4, indicating that the ionic NH4 (+) did not permeate AQP4. Molecular dynamics simulations revealed partial pore permeation events of NH3 but not of NH4 (+) and a reduced energy barrier for NH3 permeation through AQP4 compared with that of a cholesterol-containing lipid bilayer, suggesting AQP4 as a favored transmembrane route for NH3 Our data propose that AQP4 belongs to the growing list of NH3-permeable water channels.