Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr residue

Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 (⁶⁰⁰SHIFAGGYAAGYYSY⁶¹⁴). Modeling studies indicated that in OpdA the Tyr⁶⁰⁷ residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr⁶⁰⁷ by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. The kinetic parameters showed the importance of Tyr⁶⁰⁷ in OpdA catalytic activity as its substitution promoted a decrease in the k_cat/K_m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr⁶⁰⁷ residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity.     

Alternating Hemiplegia of Childhood mutations have a differential effect on Na+,K+-ATPase activity and ouabain binding

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na⁺,K⁺-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na⁺,K⁺-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K⁺/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na⁺,K⁺-ATPase. Functional impairment of Na⁺,K⁺-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.     

A functional screen provides evidence for a conserved, regulatory, juxtamembrane phosphorylation site in guanylyl cyclase a and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ~20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation--processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200-1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor (32)PO(4) co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.     

Site-specific mutagenesis on Mnemiopsin 2: Calcium coordination and substrate binding properties of new variants

We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (T_m) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol⁻¹) is considerably lower than those in the WT photoprotein (102 kcal mol⁻¹) and E179S mutant (106 kcal mol⁻¹). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively.     

Determinants of Cation Permeation and Drug Sensitivity in Predicted Transmembrane Helix 9 and Adjoining Exofacial Re-entrant Loop 5 of Na+/H+ Exchanger NHE1

Mammalian Na⁺/H⁺ exchangers (NHEs) regulate numerous physiological processes and are involved in the pathogenesis of several diseases, including tissue ischemia and reperfusion injuries, cardiac hypertrophy and failure, and cancer progression. Hence, NHEs are being targeted for pharmaceutical-based clinical therapies, but pertinent information regarding the structural elements involved in cation translocation and drug binding remains incomplete. Molecular manipulations of the prototypical NHE1 isoform have implicated several predicted membrane-spanning (M) helices, most notably M4, M9, and M11, as important determinants of cation permeation and drug sensitivity. Here, we have used substituted-cysteine accessibility mutagenesis and thiol-modifying methanethiosulfonate (MTS) reagents to further probe the involvement of evolutionarily conserved sites within M9 (residues 342–363) and the adjacent exofacial re-entrant loop 5 between M9 and M10 (EL5; residues 364–415) of a cysteine-less variant of rat NHE1 on its kinetic and pharmacological properties. MTS treatment significantly reduced the activity of mutants containing substitutions within M9 (H353C, S355C, and G356C) and EL5 (G403C and S405C). In the absence of MTS, mutants S355C, G403C, and S405C showed modest to significant decreases in their apparent affinities for Na⁺_o and/or H⁺_i. In addition, mutations Y370C and E395C within EL5, whereas failing to confer sensitivity to MTS, nevertheless, reduced the affinity for Na⁺_o, but not for H⁺_i. The Y370C mutant also exhibited higher affinity for ethylisopropylamiloride, a competitive antagonist of Na⁺_o transport. Collectively, these results further implicate helix M9 and EL5 of NHE1 as important elements involved in cation transport and inhibitor sensitivity, which may inform rational drug design.     

Site-Directed Mutagenesis of Active Site Residues Reveals Plasticity of Human Butyrylcholinesterase in Substrate and Inhibitor Interactions

Abstract: In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser¹⁹⁸, the acyl pocket Leu²⁸⁶ and adjacent Phe³²⁹ residues, and Met⁴³⁷ and Tyr⁴⁴⁰ located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and K_m values towards butyrylthiocholine and IC₅₀ values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser¹⁹⁸ by cysteine and Met⁴³⁷ by aspartate nearly abolished activity, and other mutations of Ser¹⁹⁸ completely abolished it. Tyr⁴⁴⁰ and Leu²⁸⁶ mutants remained active, but with higher K_m and IC₅₀ values. Rates of inhibition by DFP were roughly parallel to IC₅₀ values for several Leu²⁸⁶ mutants. Both K_m and IC₅₀ values increased for Leu²⁸⁶ mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe³²⁹ displayed unmodified K_m values toward butyrylthiocholine, but up to 10-fold decreased IC₅₀ values for DFP, iso-OMPA, and echothiophate. These findings add Tyr⁴⁴⁰ and Phe³²⁹ to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.     

Sodium orthovanadate associated with pharmacological doses of ascorbate causes an increased generation of ROS in tumor cells that inhibits proliferation and triggers apoptosis

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na₃VO₄) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na₃VO₄ was cytotoxic against T24 cells (EC₅₀ = 5.8 μM at 24 h), but in the presence of ascorbate (100 μM) the EC₅₀ fell to 3.3 μM. Na₃VO₄ plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na₃VO₄ did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na₃VO₄ and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na₃VO₄ alone, or combined with ascorbate, increased catalase activity, but only Na₃VO₄ plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na₃VO₄ plus ascorbate (2–3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na₃VO₄. The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na₃VO₄ in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment.     

Suppression of Conformation-Compromised Mutants of Salmonella enterica Serovar Typhimurium MelB

The crystal structure of the Na⁺-coupled melibiose permease of Salmonella enterica serovar Typhimurium (MelB_St) demonstrates that MelB is a member of the major facilitator superfamily of transporters. Arg residues at positions 295, 141, and 363 are involved in interdomain interactions at the cytoplasmic side by governing three clusters of electrostatic/polar interactions. Insertion of (one at a time) Glu, Leu, Gln, or Cys at positions R295, R141, and R363, or Lys at position R295, inhibits active transport of melibiose to a level of 2 to 20% of the value for wild-type (WT) MelB_St, with little effect on binding affinities for both sugar and Na⁺. Interestingly, a spontaneous suppressor, D35E (periplasmic end of helix I), was isolated from the R363Q MelB_St mutant. Introduction of the D35E mutation in each of the mutants at R295, R141 (except R141E), or R363 rescues melibiose transport to up to 91% of the WT value. Single-site mutations for the pair of D35 and R175 (periplasmic end of helix VI) were constructed by replacing Asp with Glu, Gln, or Cys and R175 with Gln, Asn, or Cys. All mutants with mutations at R175 are active, indicating that a positive charge at R175 is not necessary. Mutant D35E shows reduced transport; D35Q and D35C are nearly inactivated. Surprisingly, the D35Q mutation partially rescues both R141C and R295Q mutations. The data support the idea that Arg at position 295 and a positive charge at positions 141 and 363 are required for melibiose transport catalyzed by MelB_St, and their mutation inhibits conformational cycling, which is suppressed by a minor modification at the opposite side of the membrane.     

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na⁺ and Cl^?-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na⁺-binding sites and a putative Cl^?-binding site. Mutation of all residues in the two proposed Na⁺-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na⁺-binding site 1) and mutants S20A, S20G, S20G/G405S (Na⁺-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na⁺-binding site 1 caused a decrease of the Na⁺-K_0.5, whereas mutations of site 2 increased the K_0.5. Mutation of Na⁺-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates ³⁶Cl^? demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na⁺/1Cl^?-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.     

Differential mechanisms of Cantú syndrome–associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel

Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium (⁸⁶Rb⁺) efflux assays, we show that K_ATP channels formed with P429L, A475V, or C1039Y mutants enhance K_ATP activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive K_ATP channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation.     

Greater sensitivity to vanadate of rat renal relative to cardiac (Na++K+)-ATPase

Vanadate (VO₄^?3) produces a positive inotropic effect in rats and also promotes diuresis as well as natriuresis. Although the mechanism(s) of these effects is uncertain, in the kidney, VO₄^?3 may act through inhibition of (Na⁺+K⁺)-ATPase activity, whereas in the heart, other or additional mechanisms are likely. Under the assay conditions used in the present study, microsomal (Na⁺+K⁺)-ATPase activities from rat kidney cortex and medulla were inhibited to a greater extent than was left ventricular (Na⁺+K⁺)-ATPase activity over a range of VO₄^?3 concentrations. The apparent dissociation constant for left ventricular (Na⁺+K⁺)-ATPase (10.95 ± 1.26 × 10^?7M VO₄^?3) was significantly greater than that of (Na⁺+K⁺)-ATPase from the cortex (3.46±0.96×10^?7 M VO₄^?3) or the medulla (3.32±0.7×10^?7M VO₄^?3, N=6, P<.05) whereas there were no significant differences between the effects of VO₄^?3 on (Na⁺+K⁺)-ATPase from the cortex and medulla. The greater inhibition by VO₄, of (Na⁺+K⁺)-ATPase from the cortex relative to that of the left ventricle, occurred over a range of Na⁺ and K⁺ concentrations, and K⁺ enhanced the inhibition by VO₄^?3 to a greater extent for (Na⁺+K⁺)-ATPase from the cortex than the left ventricle. These results suggest that renal (Na⁺+K⁺)-ATPase is more sensitive than left ventricular (Na⁺+K⁺)-ATPase to inhibition by VO₄^?3 and would, therefore, be more likely to be modulated $\text{in}$$\text{vivo.}$     

Functional analysis of human Na~+/K~+-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes

AIM: Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine(FHM2, SHM). METHODS: cRNA of human Na+/K+-ATPase α2- and β1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/β interaction sites or in the α2-subunit's C-terminal region were investigated. Mutants were analyzed by the twoelectrode voltage-clamp(TEVC) technique on Xenopus oocytes. Stationary K+-induced Na+/K+ pump currents were measured, and the voltage dependence of apparent K+ affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na+ buffers to analyze kinetics and voltage-dependent presteady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. RESULTS: Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant dif-ferences in the voltage dependence of K+-induced currents, and analysis of the transient currents indicated that the extracellular Na+ affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K+ interaction, D999H affected the voltage dependence of K+-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na+ was reduced. CONCLUSION: The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.     

Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions

Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the α2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase α2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the α and β subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na⁺ and increase the K⁺ affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K⁺ and Na⁺ affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).     

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC₅₀ properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders.     

Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests

The insecticidal activities and specificities of the Vip3Aa proteins derived from different Bt strains are very different, although the similarities between these proteins are higher than 95%. In this study, we hypothesised that the differences in Vip3Aa11 and Vip3Aa39 C-terminal amino acids determine their differences in insecticidal activity against three Lepidoptera insects. To find the amino acid residues associated with insecticidal activity, nine different amino acid residues of Vip3Aa11 were substituted with the corresponding amino acid residues from Vip3Aa39 by site-directed mutagenesis. The toxicity of each protein was estimated by bioassays, and the results demonstrated that the mutant Y784N lost its insecticidal activity against three insects (Agrotis ipsilon, Helicoverpa armigera, and Spodoptera exigua). The insecticidal activity of S543N, I544L, and S686R against S. exigua increased 5-fold, 2.65-fold, and 8.98-fold, while the toxicity to H. armigera and A. ipsilon slightly decreased compared with that of the Vip3Aa11 protein. These findings indicate that the amino acid residues Ser⁵⁴³, Ile⁵⁴⁴, Thr⁶⁸⁵, Ser⁶⁸⁶, Arg⁷⁰⁴, Ile⁷⁸⁰, and Tyr⁷⁸⁴ may be insecticidal activity-related residues. Additionally, the trypsin activation of the four mutants indicated that all proteins can form a 62-kDa core fragment, except Y784N. A possible association between the insecticidal activity and trypsin sensitivity of Vip3A proteins is suggested.     

Cyclic GMP Inhibits a Pharmacologically Distinct Na+/H+ Exchanger Variant in Cultured Rat Astrocytes via an Extracellular Site of Action

Abstract: There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP-inducing C-type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pH_i). To examine whether this effect was due to inhibition of an Na⁺/H⁺ exchanger (NHE), we acidified cells by replacing extracellular Na⁺ by choline and examined the kinetics of the pH_i recovery that occurred on reintroduction of Na⁺ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na⁺-dependent pH_i recovery, even in the nominal absence of CO₂/HCO₃^?. This indicated that CNP inhibited the activity of an exchanger that was Na⁺-dependent, HCO₃^?-independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium-dependent pH_i recovery, but the native nucleotide was as potent as membrane-permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na⁺-dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pH_i transients have been shown to modulate several important astrocytic functions.     

Na+/H+ antiporter activity of the SOS1 gene: lifetime imaging analysis and electrophysiological studies on Arabidopsis seedlings

Based on sequence analysis, the salt overly sensitive (SOS1) gene has been suggested to function as a Na⁺/H⁺ antiporter located at the plasma membrane of plant cells, being expressed mostly in the meristem zone of the root and in the parenchyma cells surrounding the vascular tissue of the stem. In this study, we compared net H⁺ and Ca²⁺ fluxes and intracellular pH and [Ca²⁺]_cyt in the root meristem zone of Arabidopsis wild‐type (WT) and sos mutants before and after salt stress. In addition, we studied the effect of pretreatment with amiloride (an inhibitor of Na⁺/H⁺ antiporters) on net ion fluxes, intracellular pH and intracellular Ca²⁺ activity ([Ca²⁺]_cyt) in WT plants and sos1 mutants before and after salt stress. Net ion fluxes were measured using microelectrode ion flux estimation (MIFE) and intracellular pH and [Ca²⁺]_cyt using fluorescence lifetime imaging microscopy (FLIM) techniques. During the first 15 min after NaCl application, sos1 mutants showed net H⁺ efflux and intracellular alkalinization in the meristem zone, whereas sos2 and sos3 mutants and WT showed net H⁺ influx and slight intracellular acidification in the meristem zone. Treatment with amiloride led to intracellular acidification and lower net H⁺ flux in WT plants and to a decrease in intracellular Ca²⁺ in WT and sos1 plants. WT plants pretreated with amiloride did not show positive net H⁺ flux and intracellular acidification. After NaCl application, internal pH shifted to higher values in WT and sos1 plants. However, absolute values of H⁺ fluxes were higher and internal pH values were lower in WT plants pretreated with amiloride compared with sos1 mutants. Therefore, the SOS1 transporter is involved in H⁺ influx into the meristem zone of Arabidopsis roots, or it may function as a Na⁺/H⁺ antiporter. Amiloride affects SOS1 and other Na⁺/H⁺ antiporters in plant cells because of its ability to decrease the H⁺ gradient across the plasma membrane.     

Adenosine A2A receptor blocks the A 1 receptor inhibition of renal Na + transport and oxygen consumption

A high renal oxygen (O₂) need is primarily associated with the renal tubular O₂ consumption (VO₂) necessary for a high rate of sodium (Na⁺) transport. Limited O₂ availability leads to increased levels of adenosine, which regulates the kidney via activation of both A₁ and A_2A adenosine receptors (A1R and A2AR, respectively). The relative contributions of A1R and A2AR to the regulation of renal Na⁺ transport and VO₂ have not been determined. We demonstrated that A1R activation has a dose-dependent biphasic effect on both renal Na⁺/H⁺ exchanger-3 (NHE3), a major player in Na⁺ transport, and VO₂. Here, we report concentration-dependent effects of adenosine: less than 5 × 10⁻⁷ M adenosine-stimulated NHE3 activity; between 5 × 10⁻⁷ M and 10⁻⁵ M adenosine-inhibited NHE3 activity; and greater than 10⁻⁵ M adenosine reversed the change in NHE3 activity (returned to baseline). A1R activation mediated the activation and inhibition of NHE3 activity, whereas 10⁻⁴ M adenosine had no effect on the NHE3 activity due to A2AR activation. The following occurred when A1R and A2AR were activated: (a) Blockade of the A2AR receptor restored the NHE3 inhibition mediated by A1R activation, (b) the NHE-dependent effect on VO₂ mediated by A1R activation became NHE independent, and (c) A2AR bound to A1R. In summary, A1R affects VO₂ via NHE-dependent mechanisms, whereas A2AR acts via NHE-independent mechanisms. When both A1R and A2AR are activated, the A2AR effect on NHE3 and VO₂ predominates, possibly via an A1R–A2AR protein interaction. A2AR–A1R heterodimerization is proposed as the molecular mechanism enabling the NHE-independent control of renal VO₂.     

Phosphatidic acid regulates signal output by G protein coupled receptors through direct interaction with phospholipase C-β1

Phosphatidic acid (PA), generated downstream of monomeric Rho GTPases via phospholipase D (PLD) and additionally by diacylglycerol kinases (DGK), both stimulates phospholipase C-β₁ (PLC-β₁) and potentiates stimulation of PLC-β₁ activity by Gα_q in vitro. PA is a potential candidate for integrating signaling by monomeric and heterotrimeric G proteins to regulate signal output by G protein coupled receptors (GPCR), and we have sought to understand the mechanisms involved. We previously identified the region spanning residues 944–957, lying within the PLC-β₁ C-terminus αA helix and flexible loop of the Gα_q binding domain, as required for stimulation of lipase activity by PA in vitro. Regulation by PA does not require residues essential for stimulation by Gα_q or GTPase activating activity. The present studies evaluated shorter alanine/glycine replacement mutants and finally point mutations to identify Tyr⁹⁵² and Ile⁹⁵⁵ as key determinants for regulation by PA, assessed by both in vitro enzymatic and cell-based co-transfection assays. Replacement of Tyr⁹⁵² and Ile⁹⁵⁵, PLC-β₁ (Y952G/I955G), results in an 85% loss in stimulation by PA relative to WT-PLC-β₁ in vitro. COS 7 cells co-transfected with PLC-β₁ (Y952G/I955G) demonstrate a 10-fold increase in the EC₅₀ for stimulation and a 60% decrease in maximum stimulation by carbachol via Gα_q linked m1 muscarinic receptors, relative to cells co-transfected with WT-PLC-β₁ but otherwise similar conditions. Residues required for regulation by PA are not essential for stimulation by G protein subunits. WT-PLC-β₁ and PLC-β₁(Y952G/I955G) activity is increased comparably by co-transfection with Gα_q and neither is markedly affected by co-transfection with Gβ₁γ₂. Inhibiting PLD-generated PA production by 1-butanol has little effect on maximum stimulation, but shifts the EC₅₀ for agonist stimulation of WT-PLC-β₁ by 10-fold, producing a phenotype similar to PLC-β₁ (Y952G/I955G) with respect to agonist potency. 1-Butanol is without effect on carbachol stimulated PLC activity in cells co-transfected with either PLC-β₁(Y952G/I955G) or on endogenous PLC activity, indicating that regulation by PA requires direct interaction with the PLC-β₁ PA-binding region. These data show that endogenous PA regulates signal output by Gα_q-linked GPCRs in transfected cells directly through PLC-β₁. Gα_q and PA may co-ordinate to regulate signaling. Regulation by PA may constitute part of a mechanism that routes receptor signaling to specific PLC isoforms.