Extracellular Proton Concentration Research Articles

Microalgal photosynthesis induces alkalization of aquatic environment as a result of H+ uptake independently from CO2 concentration – New perspectives for environmental applications

The photosynthetic process in microalgae and the extracellular proton environment interact with each other. The photosynthetic process in microalgae induces a pH increase in the aquatic environment as a result of cellular protons uptake rather than as an effect of CO2 consumption. The photosynthetic water photolysis and the reduction/oxidation cycle of the plastoquinone pool provide lumen with protons. Weak bases act as “permeant buffers” in lumen during the photosynthetic procedure, converting the ΔpH to Δψ. This is possibly the main reason for continuous light-driven proton uptake from the aquatic environment through cytosol and stroma, into the lumen. The proton uptake rate and, therefore, the microalgal growth is proportional to the light intensity, cell concentration, and extracellular proton concentration. The low pH in microalgae cultures, without limitation factors related to light and nutrients, strongly induces photosynthesis (and proton uptake) and, consequently, growth. In contrast, the mitochondrial respiratory process, in the absence of photosynthetic activity, does not substantially alter the culture pH. Only after intensification of the respiratory process, using exogenous glucose supply leads to significantly reduced pH values in the culture medium, almost exclusively through proton output. Enhanced dissolution of atmospheric CO2 in water causes the phenomenon of ocean acidification, which prevents the process of calcification, a significant process for numerous phytoplankton and zooplankton organisms, as well for corals. The proposed interaction between microalgal photosynthetic activity and proton concentration in the aquatic environment, independently from the CO2 concentration, paves the way for new innovative management strategies for reversing the ocean acidification.

Functional Role of Microbiota‐derived Metabolites in the GPCR‐mediated Regulation of Intestinal Wound Healing and Barrier Function

The mammalian intestine houses a complex and abundant ensemble of commensal bacteria, which are in constant cross‐talk with the intestinal epithelial cells, and thereby, ensure gut homeostasis, development, permeability, and repair. Wound regeneration is a complex but coordinated cellular events to repair injured gut mucosa, which involves both cellular proliferation and migration. Gut barrier function is central to the health and is critical in a wide variety of diseases including inflammatory bowel diseases (IBD). Our recent metagenomics study discovered a consortium of novel gut mucosa‐associated proregenerative symbiotic bacteria, which preferentially enrich in and colonize the injured mucosa to augment epithelial repair. The resident microbiota of the gut is endowed with over a hundred times more genes than those in our genome. However, the microbiota's more than nine million genes, and their metabolic functions remained mostly unrecognized. We performed an unbiased screening analysis by mass spectrometry‐based metabolomic profiling of intact and wounded mucosa in germ‐free and conventional mice. Unlike the germ‐free animals, the colon of the conventional mice demonstrated increased amounts of acidic microbial metabolites, which established an extracellular pH gradient with more acidic pH at the apex of the colonic crypt. We found that the murine and human intestinal epithelial cells express the proton‐sensing G‐protein coupled‐receptor GPCR68. Intriguingly, the murine colonic contents of the conventional mouse or a specific metabolite, 2‐hydroxy‐4‐methylpentanoic acid (HMP) decreases the extracellular pH to modulate claudin‐4 dependent paracellular permeability and to regulate the proliferation of intestinal epithelial cells in a GPCR68‐dependent manner. Importantly, our findings highlighted that the HMP‐stimulated intestinal epithelial wound closure requires GPR68. Collectively, our data underscore the significance of intestinal metabolites in modulating the extracellular proton concentration in the gut mucosal microenvironment. Furthermore, our findings demonstrate that the GPCR68 functions as the sensor of microbially modulated proton gradient that is potentially involved in the maintenance of gut mucosal homeostasis, wound healing, and restoration of the intestinal barrier function.Support or Funding InformationK01DK114391 (National Institute of Diabetes and Digestive and Kidney Diseases), R01DK089763 (National Institute of Diabetes and Digestive and Kidney Diseases), R01AI064462 (National Institute of Allergy and Infectious Diseases)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Expression of proton-sensing G-protein-coupled receptors in selected skin tumors.

In humans, there are four known proton-sensing G-Protein-coupled receptors (pH-GPCRs): GPR4 (GPR19), TDAG8 (GPR65, T-cell death-associated gene 8), OGR1 (GPR68, ovarian cancer GPCR1) and G2A (GPR132, G2 accumulation protein). They are known to be involved in sensing changes of extracellular proton concentrations in the acidic microenvironment of tumors, which leads to altered cell proliferation, migration, metastasis, immune cell function and inflammation. However, little is known about the expression of pH-GPCRs in the skin and especially skin cancers. We studied the expression of pH-GPCRs in selected skin cancers, that is Merkel cell carcinoma (MCC), dermatofibrosarcoma protuberans (DFSP), atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS). We did immunohistochemistry and immunofluorescence to analyse the expression of GPR4, TDAG8, OGR1 and G2A using paraffin-embedded tissue samples (n=4, exceptions: PDS GPR4/GPR65 n=5, AFX GPR132 n=3) from patients suffering from MCC, DFSP, AFX and PDS. (a) GPR4 was expressed on all AFX and PDS specimens. All AFX and MCC showed a positive expression of G2A. All PDS exhibited a strong positive expression of G2A. (b) MCCs neither expressed GPR4 nor TDAG8. All DFSP showed no expression of TDAG8. (c) For any other combination of GPCR and skin disease, we found positive/negative mixed results. These are the first results on pH-GPCRs in selected skin cancers. We provide evidence that these GPCRs are differentially expressed on the various types of skin cancers and that they can potentially be addressed as a therapeutic target in extensive disease.

Protons as Messengers of Intercellular Communication in the Nervous System.

In this review, evidence demonstrating that protons (H+) constitute a complex, regulated intercellular signaling mechanisms are presented. Given that pH is a strictly regulated variable in multicellular organisms, localized extracellular pH changes may constitute significant signals of cellular processes that occur in a cell or a group of cells. Several studies have demonstrated that the low pH of synaptic vesicles implies that neurotransmitter release is always accompanied by the co-release of H+ into the synaptic cleft, leading to transient extracellular pH shifts. Also, evidence has accumulated indicating that extracellular H+ concentration regulation is complex and implies a source of protons in a network of transporters, ion exchangers, and buffer capacity of the media that may finally establish the extracellular proton concentration. The activation of membrane transporters, increased production of CO2 and of metabolites, such as lactate, produce significant extracellular pH shifts in nano- and micro-domains in the central nervous system (CNS), constituting a reliable signal for intercellular communication. The acid sensing ion channels (ASIC) function as specific signal sensors of proton signaling mechanism, detecting subtle variations of extracellular H+ in a range varying from pH 5 to 8. The main question in relation to this signaling system is whether it is only synaptically restricted, or a volume modulator of neuron excitability. This signaling system may have evolved from a metabolic activity detection mechanism to a highly localized extracellular proton dependent communication mechanism. In this study, evidence showing the mechanisms of regulation of extracellular pH shifts and of the ASICs and its function in modulating the excitability in various systems is reviewed, including data and its role in synaptic neurotransmission, volume transmission and even segregated neurotransmission, leading to a reliable extracellular signaling mechanism.

On a coupled SDE-PDE system modeling acid-mediated tumor invasion

We propose and analyze a multiscale model for acid-mediated tumor invasion accounting for stochastic effects on the subcellular level. The setting involves a PDE of reaction-diffusion-taxis type describing the evolution of the tumor cell density, the movement being directed towards pH gradients in the local microenvironment, which is coupled to a PDE-SDE system characterizing the dynamics of extracellular and intracellular proton concentrations, respectively. The global well-posedness of the model is shown and numerical simulations are performed in order to illustrate the solution behavior.

A selectivity filter at the intracellular end of the acid-sensing ion channel pore.

Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the "GAS belt" in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.

Extracellular Proton Concentrations Impacts LN229 Glioblastoma Tumor Cell Fate via Differential Modulation of Surface Lipids.

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer with marginal survival rates. GBM extracellular acidosis can profoundly impact its cell fate heterogeneities and progression. However, the molecules and mechanisms that enable GBM tumor cells acid adaptation and consequent cell fate competencies are weakly understood. Since extracellular proton concentrations (pHe) directly intercept the tumor cell plasma membrane, surface lipids must play a crucial role in pHe-dependent tumor cell fate dynamics. Hence, a more detailed insight into the finely tuned pH-dependent modulation of surface lipids is required to generate strategies that can inhibit or surpass tumor cell acid adaptation, thereby forcing the eradication of heterogeneous oncogenic niches, without affecting the normal cells. By using image-based single cell analysis and physicochemical techniques, we made a small-scale survey of the effects of pH ranges (physiological: pHe 7.4, low: 6.2, and very low: 3.4) on LN229 glioblastoma cell line surface remodeling and analyzed the consequent cell fate heterogeneities with relevant molecular targets and behavioral assays. Through this basic study, we uncovered that the extracellular proton concentration (1) modulates surface cholesterol-driven cell fate dynamics and (2) induces 'differential clustering' of surface resident GM3 glycosphingolipid which together coordinates the proliferation, migration, survival, and death reprogramming via distinct effects on the tumor cell biomechanical homeostasis. A novel synergy of anti-GM3 antibody and cyclophilin A inhibitor was found to mimic the very low pHe-mediated GM3 supraclustered conformation that elevated the surface rigidity and mechano-remodeled the tumor cell into a differentiated phenotype which eventually succumbed to the anoikis type of cell death, thereby eradicating the tumorigenic niches. This work presents an initial insight into the physicochemical capacities of extracellular protons in the generation of glioblastoma tumor cell heterogeneities and cell death via the crucial interplay of surface lipids and their conformational changes. Hence, monitoring of proton-cholesterol-GM3 correlations in vivo through diagnostic imaging and in vitro in clinical samples may assist better tumor staging and prognosis. The emerged insights have further led to the translation of a 'pH-dependent mechanisms of oncogenesis control' into the surface targeted anti-GBM therapeutics.

Proton-sensing G protein-coupled receptors as regulators of cell proliferation and migration during tumor growth and wound healing.

Dysregulation of pH is a feature of both tumor growth and tissue repair. In tumors, microenvironmental changes, like in lactate metabolism, lead to altered intra- and extracellular pH (pHi , pHe ) and vice versa. In wounds, barrier disruption results in extensive variations in pHe on the wound surface. It is known that altered extracellular proton concentrations have a major impact on cell turnover and migration as well as on the metabolic activity of cells involved in tumor spread and wound closure. The proton-sensing G protein-coupled receptors (GPCRs) GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A) are activated via a decrease in pHe and transduce this signal to molecular intracellular pathways. Based on the current knowledge, we speculate on the role of proton-sensing GPCRs in wound healing and on their potential as mechanistic linkers of tumor growth and tissue repair.

Extracellular protons enable activation of the calcium-dependent chloride channel TMEM16A.

The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca2+ -dependent Cl- channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+ ]o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H+ ]o from 10-10 to 10-5.5 m caused a progressive increase in the chloride current (ICl ) that is described by titration of a protonatable site with pK=7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+ ]i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10-5.5 and 10-9.0 m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10-5.5 m, but 10-9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+ ]i .

A nonlocal sample dependence SDE-PDE system modeling proton dynamics in a tumor

A nonlocal stochastic model for intra- and extracellular proton dynamics in a tumor is proposed.The intracellular dynamics is governed by an SDE coupled to a reaction-diffusionequation for the extracellular proton concentration on the macroscale. In a more general contextthe existence and uniqueness of solutions for local and nonlocalSDE-PDE systems are established allowing, in particular, to analyze the proton dynamics model, bothin its local version and in the case with nonlocal path dependence.Numerical simulations are performedto illustrate the behavior of solutions, providing some insights into the effects of randomness on tumor acidity.

Acid-Sensing Ion Channels Expression, Identity and Role in the Excitability of the Cochlear Afferent Neurons.

Acid-sensing ion channels (ASICs) are activated by an increase in the extracellular proton concentration. There are four genes (ASIC1-4) that encode six subunits, and they are involved in diverse neuronal functions, such as mechanosensation, learning and memory, nociception, and modulation of retinal function. In this study, we characterize the ASIC currents of spiral ganglion neurons (SGNs). These ASIC currents are primarily carried by Na+, exhibit fast activation and desensitization, display a pH50 of 6.2 and are blocked by amiloride, indicating that these are ASIC currents. The ASIC currents were further characterized using several pharmacological tools. Gadolinium and acetylsalicylic acid reduced these currents, and FMRFamide, zinc (at high concentrations) and N,N,N’,N’–tetrakis-(2-piridilmetil)-ethylenediamine increased them, indicating that functional ASICs are composed of the subunits ASIC1, ASIC2, and ASIC3. Neomycin and streptomycin reduced the desensitization rate of the ASIC current in SGNs, indicating that ASICs may contribute to the ototoxic action of aminoglycosides. RT-PCR of the spiral ganglion revealed significant expression of all ASIC subunits. By immunohistochemistry the expression of the ASIC1a, ASIC2a, ASIC2b, and ASIC3 subunits was detected in SGNs. Although only a few SGNs exhibited action potential firing in response to an acidic stimulus, protons in the extracellular solution modulated SGN activity during sinusoidal stimulation. Our results show that protons modulate the excitability of SGNs via ASICs.

The Verapamil Transporter Expressed in Human Alveolar Epithelial Cells (A549) Does Not Interact with β2-Receptor Agonists

Affinity of different organs for verapamil is highly variable and organ-specific. For example, the drug exhibits high levels of accumulation in lung tissues. A transporter recognising verapamil as a substrate has previously been identified in human retinal pigment epithelial (RPE) and in rat retinal capillary endothelial (TR-iBRB2) cells. This transporter is distinct from any of the cloned organic cation transporters. Therefore, we hypothesised that the verapamil transporter is also functionally expressed in the human respiratory mucosa. Moreover, we tested the hypothesis that this transporter interacts with pulmonary administered cationic drugs such as β2-agonists. The uptake of [(3)H]verapamil was studied in A549 human alveolar epithelial cell monolayers at different times and concentrations. The influence of extracellular proton concentration and various organic cations on verapamil uptake was determined. Verapamil uptake into A549 cells was time- and concentration-dependent, sensitive to pH and had a Km value of 39.8 ± 8.2 µM. Verapamil uptake was also sensitive to inhibition by amantadine, quinidine and pyrilamine, but insensitive to other typical modulators of organic cation and choline transporters. Whilst we demonstrated functional activity of the elusive verapamil transporter at the lung epithelium, our data suggest that this transporter does not interact with β2-agonists at therapeutic concentrations.

Plasma Membrane-Associated Glycohydrolases Activation by Extracellular Acidification due to Proton Exchangers

In this paper, we show that the pH optimum for the plasma membrane (PM)-associated activity of four glycohydrolases (conduritol B epoxide sensitive β-glucosidase, β-glucosidase GBA2, β-hexosaminidase and β-galactosidase) measured on intact cells is acidic. Moreover, we show that drugs able to modify the efflux of protons across the PM, thus locally affecting the extracellular proton concentration close to the PM, are able to modulate the activities of these enzymes. These data strongly suggest that pH-dependent modulation of PM-associated glycohydrolases activities could be an effective way to locally modulate the cell surface glycoconjugate composition.

Proton feedback mediates the cascade of color-opponent signals onto H3 horizontal cells in goldfish retina

It has been postulated that horizontal cells (HCs) send feedback signals onto cones via a proton feedback mechanism, which generates the center-surround receptive field of bipolar cells, and color-opponent signals in many non-mammalian vertebrates. Here we used a strong pH buffer, HEPES, to reduce extracellular proton concentration changes and so determine whether protons mediate color-opponent signals in goldfish H3 (triphasic) HCs. Superfusion with 10mM HEPES-fortified saline elicited depolarization of H3 HCs’ dark membrane potential and enhanced hyperpolarizing responses to blue stimuli, but suppressed both depolarization by yellow and orange and hyperpolarization by red stimuli. The response components suppressed by HEPES resembled the inverse of spectral responses of H2 (biphasic) HCs. These results are consistent with the Stell–Lightfoot cascade model, in which the HEPES-suppressed component of H3 HCs was calculated using light responses recorded experimentally in H1 (monophasic) and H2 HCs. Selective suppression of long- or long-+middle-wavelength cone signals by long-wavelength background enhanced the responses to short-wavelength stimuli. These results suggest that HEPES inhibited color opponent signals in H3 HCs, in which the source of opponent-color signals is primarily a feedback from H2 HCs and partly from H1 HCs onto short-wavelength cones, probably mediated by protons.

Drug-Induced hERG Current Enhancement is Modulated by Extracellular Proton and Potassium

Acid-base disturbances and hypokalemia or hyperkalemia are frequently encountered in a variety of clinical scenarios. Extracellular proton and potassium concentrations are important modulators of hERG channel functions and biophysical properties such as deactivation and inactivation. Slowing deactivation and inactivation kinetics of hERG channel are two important components for drug-induced hERG current enhancement. This study examined if drug-induced hERG current enhancement is affected by acid-base and electrolyte aberrations at physiological temperature (37° C). Whole-cell voltage clamp technique was used to measure hERG current (step-ramp protocol: initial 1-sec depolarization step to 0 mV from −80 mV (VH) followed by a 2-sec repolarization ramp back to VH) and a typical hERG activator A-935142 was used as a tool drug. Drug-induced hERG current enhancement was potentiated by extracellular acidification (% increase for step current: 132 ± 5 vs. 58.4 ± 4.2, pH 6.8 vs. 7.4, P < 0.01; % increase for tail current: 115 ± 3 vs. 43.2 ± 3.2, P < 0.01). In contrast, alkalosis (pH 8.4) weakened the drug-induced hERG current enhancement (% increase for step: 30.3 ± 3.1 vs. 58.4 ± 4.2, pH 8.4 vs 7.4, P < 0.01; % increase for tail: 21.9 ± 2.3 vs. 43.2 ± 3.2, P < 0.01). The measured bath drug concentrations were the same at normal pH, acidosis, and alkalosis. Hypokalemia (1 mM K+) did not affect drug-induced hERG current enhancement but hyperkalemia (10 mM K+) attenuated the drug-induced hERG current enhancement (increase for step: 41.1 ± 1.6% vs. 58.4 ± 4.2%, 10 mM vs. 5 mM K+, P < 0.01; increase for tail: 31.5 ± 1.3 % vs. 43.2 ± 3.2 %, P < 0.05). These results demonstrate that hERG current enhancement by A-935142 is modulated by extracellular proton concentrations and hyperkalemia.

Long-Term Adaptation of Bacillus subtilis 168 to Extreme pH Affects Chemical and Physical Properties of the Cellular Membrane

We characterized physical and chemical properties of cell-membrane fragments from Bacillus subtilis 168 (trpC2) grown at pH 5.0, 7.0 and 8.5. Effects of long-term bacterial adaptation reflected in growth rates and in changes of the membrane lipid composition were correlated with lipid order and dynamics using time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. We demonstrate that the pH adaptation results in a modification of a fatty acid content of cellular membranes that significantly influences both the lipid-chain order and dynamics. For cultivation at acidic conditions, the lipid order increases and membrane dynamics decreases compared to pH 7.0. This results in rigid and ordered membranes. Cultivation at pH 8.5 causes slight membrane disordering. Instant pH changes induce qualitatively similar but smaller effects. Proton flux measurements performed on intact cells adapted to both pH 5.0 and 8.5 revealed lower cell-membrane permeability compared to bacteria cultivated at pH optimum. Our results indicate that both acidic and alkalic pH stress represent a permanent challenge for B. subtilis to keep a functional membrane state. The documented adaptation-induced adjustments of membrane properties could be an important part of mechanisms maintaining an optimal intracellular pH at a wide range of extracellular proton concentrations.

Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl(-)/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3(-)/H+ antiporter atClCa, led to coupled NO3(-)/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3(-)/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of approximately 7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.

Identification of the amino acid residues in the extracellular domain of rat P2X7 receptor involved in functional inhibition by acidic pH

P2X(7), receptors are potently inhibited by extracellular acidification. The underlying molecular basis remains unknown. This study aimed to examine the role of extracellular histidine, lysine, aspartic acid and glutamic acid residues in the functional inhibition of rat P2X(7) receptors by acidic pH. We introduced point mutations into rat P2X(7) receptor by site-directed mutagenesis, expressed wild type (WT) and mutant receptors in human embryonic kidney (HEK293) cells and, using patch clamp recording, characterized the effects of acidic pH on BzATP [2'-3'O-(4-benzoylbenzoyl) adenosine 5'-triphosphate]-evoked ionic currents. Reducing extracellular pH, that is, increasing extracellular proton concentrations, inhibited BzATP-evoked currents in cells expressing WT P2X(7) receptors, with IC(50) value (half-maximal antagonist or inhibitor concentration) for protons of 0.2 mumol.L(-1). The major effect of acidification was suppression of the maximal current response without altering the agonist sensitivity. five residues in the receptor extracellular domain (His(85), Lys(110), Lys(137), Asp(197) and His(219)) were mutated to alanine and current inhibition by protons assessed. Compared with WT, the H85A, H219A, K137A mutants were two- to threefold more sensitive, whereas the K110A and D197A mutants were 2.5- and 9-fold less sensitive. Double-alanine substitution of Lys(110) and Asp(197) resulted in 23-fold decreased sensitivity to inhibition by protons. Furthermore, charge neutralization (K110M, K110F, D197N and D197F), but not charge conserving mutation (K110R and D197E), attenuated the inhibition of currents by protons. Functional inhibition of rat P2X(7) receptors by acidic pH was variably affected by the extra-cellular His(85), Lys(110), Lys(137), Asp(197) and His(219) residues, with the Asp(197) residue being most critical for this inhibition.

Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines

Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain. Using organotypic hippocampal slices and biolistic transfection, we found the acid-sensing ion channel 1a (ASIC1a), localized in dendritic spines where it functioned as a proton receptor. ASIC1a also affected the density of spines, the postsynaptic site of most excitatory synapses. Decreasing ASIC1a reduced the number of spines, whereas overexpressing ASIC1a had the opposite effect. Ca(2+)-mediated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) signaling was probably responsible, because acid evoked an ASIC1a-dependent elevation of spine intracellular Ca(2+) concentration, and reducing or increasing ASIC1a levels caused parallel changes in CaMKII phosphorylation in vivo. Moreover, inhibiting CaMKII prevented ASIC1a from increasing spine density. These data indicate that ASIC1a functions as a postsynaptic proton receptor that influences intracellular Ca(2+) concentration and CaMKII phosphorylation and thereby the density of dendritic spines. The results provide insight into how protons influence brain function and how they may contribute to pathophysiology.