Channel surfing uncovers a dual‐use transporter

Beyond their roles in generating nerve impulses, ion channels are important for many facets of cell biology, including swelling and osmotic stress responses. Sabirov and colleagues now report that the prostaglandin transporter SLCO2A1 is a central component of the ubiquitous Maxi‐Cl anion channel. These findings add to a growing number of transporters displaying ion channel activity and not only provide a molecular handle for future Maxi‐Cl physiological and biophysical studies, but also underscore general questions about the principles that underlie such transporter/channel dual‐use behavior.     

AtALMT12 represents an R‐type anion channel required for stomatal movement in Arabidopsis guard cells

Stomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells. Anion efflux from guard cells involves slow (S‐type) and rapid (R‐type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage‐independent anion channel component in guard cells. In contrast, the R‐type channel still awaits identification. Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R‐type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane. Plants lacking AtALMT12 are impaired in dark‐ and CO₂‐induced stomatal closure, as well as in response to the drought‐stress hormone abscisic acid. Patch‐clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R‐type currents compared with wild‐type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage‐dependent anion currents reminiscent to R‐type channels could be activated. In line with the features of the R‐type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials. R‐Type channels, like voltage‐dependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long‐term anion and K⁺ efflux via SLAC1 and GORK, respectively.     

Open stomata 1 (OST1) kinase controls R–type anion channel QUAC1 in Arabidopsis guard cells

Under drought stress, the stress hormone ABA addresses the SnR kinase OST1 via its cytosolic receptor and the protein phosphatase ABI1. Upon activation, OST1 phosphorylates the guard cell S–type anion channel SLAC1. Arabidopsis ABI1 and OST1 loss‐of‐function mutants are characterized by an extreme wilting 'open stomata′ phenotype. Given the fact that guard cells express both SLAC‐ and R–/QUAC‐type anion channels, we questioned whether OST1, besides SLAC1, also controls the QUAC1 channel. In other words, are ABI1/OST1 defects preventing both of the guard cell anion channel types from operating properly in terms of stomatal closure? The activation of the R–/QUAC‐type anion channel by ABA signaling kinase OST1 and phosphatase ABI1 was analyzed in two experimental systems: Arabidopsis guard cells and the plant cell‐free background of Xenopus oocytes. Patch‐clamp studies on guard cells show that ABA activates R–/QUAC‐type currents of wild‐type plants, but to a much lesser extent in those of abi1–1 and ost1–2 mutants. In the oocyte system the co‐expression of QUAC1 and OST1 resulted in a pronounced activation of the R–type anion channel. These studies indicate that OST1 is addressing both S–/SLAC‐ and R–/QUAC‐type guard cell anion channels, and explain why the ost1–2 mutant is much more sensitive to drought than single slac1 or quac1 mutants.     

Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive cl(-) channels.

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.     

Vacuolar malate uptake is mediated by an anion-selective inward rectifier

Electrophysiological studies using the patch‐clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current–voltage relations of the tonoplast were dominated by a strongly inward‐rectifying anion‐selective channel that was active at cytoplasmic‐side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage‐dependent gating of a 3‐pS malate‐selective ion channel, which showed a half‐maximal open probability at ?43 mV. Also, the time‐averaged unitary currents following a step to a negative voltage corresponded to the time‐dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion‐selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent K_m of 2.5 mm malate^2? activity), a selectivity sequence of fumarate^2? > malate^2? > Cl^? > maleate^2– ≈ citrate^3–, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 µm niflumate reversibly blocked the activity of the 3‐pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 µm^?2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3‐pS, inward‐rectifying, anion‐selective channel represents the principal pathway for malate influx into the vacuole.     

Two novel mutations in the SLCO2A1 gene in a Chinese patient with primary hypertrophic osteoarthropathy

Primary hypertrophic osteoarthropathy (PHO) is a rare monogenetic disease characterized by digital clubbing, periostosis and pachydermia. Mutations in the 15-hydroxy-prostaglandin dehydrogenase (HPGD) gene and solute carrier organic anion transporter family member 2A1 (SLCO2A1) gene have been shown to be associated with PHO. Here, we described clinical characteristics in a Chinese patient with PHO, and identified two novel mutations in SLCO2A1: a heterozygous guanine-to-thymidine transition at the invariant − 1 position of the acceptor site of intron 2 (c.235-1G > T) and a heterozygous missense mutation p.Pro219Leu (c.656C > T) in exon 5.     

Exome sequencing identifies SLCO2A1 mutations as a cause of primary hypertrophic osteoarthropathy

By using whole-exome sequencing, we identified a homozygous guanine-to-adenine transition at the invariant -1 position of the acceptor site of intron 1 (c.97-1G>A) in solute carrier organic anion transporter family member 2A1 (SLCO2A1), which encodes a prostaglandin transporter protein, as the causative mutation in a single individual with primary hypertrophic osteoarthropathy (PHO) from a consanguineous family. In two other affected individuals with PHO from two unrelated nonconsanguineous families, we identified two different compound heterozygous mutations by using Sanger sequencing. These findings confirm that SLCO2A1 mutations inactivate prostaglandin E(2) (PGE(2)) transport, and they indicate that mutations in SLCO2A1 are the pathogenic cause of PHO. Moreover, this study might also help to explain the cause of secondary hypertrophic osteoarthropathy.     

Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia

Airway epithelia are confronted with distinct signals emanating from the luminal and/or serosal environments. This study tested whether airway epithelia exhibit polarized intracellular free calcium (Ca(2+)(i)) and anion secretory responses to 5' triphosphate nucleotides (ATP/UTP), which may be released across both barriers of these epithelia. In both normal and cystic fibrosis (CF) airway epithelia, mucosal exposure to ATP/UTP increased Ca(2+)(i) and anion secretion, but both responses were greater in magnitude for CF epithelia. In CF epithelia, the mucosal nucleotide-induced response was mediated exclusively via Ca(2+)(i) interacting with a Ca(2+)-activated Cl(-) channel (CaCC). In normal airway epithelia (but not CF), nucleotides stimulated a component of anion secretion via a chelerythrine-sensitive, Ca(2+)-independent PKC activation of cystic fibrosis transmembrane conductance regulator. In normal and CF airway epithelia, serosally applied ATP or UTP were equally effective in mobilizing Ca(2+)(i). However, serosally applied nucleotides failed to induce anion transport in CF epithelia, whereas a PKC-regulated anion secretory response was detected in normal airway epithelia. We conclude that (1) in normal nasal epithelium, apical/basolateral purinergic receptor activation by ATP/UTP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive (PKC-mediated) anion conductances; (2) in CF airway epithelia, the mucosal ATP/UTP-dependent anion secretory response is mediated exclusively via Ca(2+)(i); and (3) Ca(2+)(i) regulation of the Ca(2+)-sensitive anion conductance (via CaCC) is compartmentalized in both CF and normal airway epithelia, with basolaterally released Ca(2+)(i) failing to activate CaCC in both epithelia.     

Tailoring Triple‐Anion Perovskite Material for Indoor Light Harvesting with Restrained Halide Segregation and Record High Efficiency Beyond 36%

Indoor photovoltaics are promising to enable self‐powered electronic devices for the Internet of Things. Here, reported is a triple‐anion CH₃NH₃PbI_2?xBrCl_x perovskite film, of which the bandgap is specially designed for indoor light harvesting to achieve a record high efficiency of 36.2% with distinctive high open circuit voltage (V_oc) of 1.028 V under standard 1000 lux fluorescent light. The involvement of both bromide and chloride suppresses the trap‐states and nonradiative recombination loss, exhibiting a remarkable ideality factor of 1.097. The introduction of chloride successfully restrains the halide segregation of iodide and bromide, stabilizing the triple‐anion perovskite film. The devices show an excellent long‐term performance, sustaining over 95% of original efficiency under continuous light soaking over 2000 h. These findings show the importance and potential of I/Br/Cl triple‐anion perovskite with tailored bandgap and suppressed trap‐states in stable and efficient indoor light recycling.     

Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

A Hereditary Enteropathy Caused by Mutations in the SLCO2A1 Gene,Encoding a Prostaglandin Transporter

Previously, we proposed a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine as chronic nonspecific multiple ulcers of the small intestine (CNSU). By whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, we found four candidate mutations in the SLCO2A1 gene, encoding a prostaglandin transporter. The pathogenicity of the mutations was supported by segregation analysis and genotyping data in controls. By Sanger sequencing of the coding regions, 11 of 12 other CNSU patients and 2 of 603 patients with a diagnosis of Crohn’s disease were found to have homozygous or compound heterozygous SLCO2A1 mutations. In total, we identified recessive SLCO2A1 mutations located at seven sites. Using RT-PCR, we demonstrated that the identified splice-site mutations altered the RNA splicing, and introduced a premature stop codon. Tracer prostaglandin E2 uptake analysis showed that the mutant SLCO2A1 protein for each mutation exhibited impaired prostaglandin transport. Immunohistochemistry and immunofluorescence analyses revealed that SLCO2A1 protein was expressed on the cellular membrane of vascular endothelial cells in the small intestinal mucosa in control subjects, but was not detected in affected individuals. These findings indicate that loss-of-function mutations in the SLCO2A1 gene encoding a prostaglandin transporter cause the hereditary enteropathy CNSU. We suggest a more appropriate nomenclature of “chronic enteropathy associated with SLCO2A1 gene” (CEAS).     

Ca<Superscript>2+</Superscript> current of frog vestibular hair cells is modulated by intracellular ATP but not by long-lasting depolarisation

Some aspects of Ca²⁺ channel modulation in hair cells isolated from semicircular canals of the frog (Rana esculenta) have been investigated using the whole-cell technique and intra and extracellular solutions designed to modify the basic properties of the Ca²⁺ macrocurrent. With 1 mM ATP in the pipette solution, about 60% of the recorded cells displayed a Ca²⁺ current constituted by a mix of an L and a drug-resistant (R2) component; the remaining 40% exhibited an additional drug-resistant fraction (R1), which inactivated in a Ca-dependent manner. If the pipette ATP was raised to 10 mM, cells exhibiting the R1 current fraction displayed an increase of both the R1 and L components by ∼280 and ∼70%, respectively, while cells initially lacking R1 showed a similar increase in the L component with R1 becoming apparent and raising up to a mean amplitude of ∼44 pA. In both cell types the R2 current fraction was negligibly affect by ATP. The current run-up was unaffected by cyclic nucleotides, and was not triggered by 10 mM ATPγS, ADP, AMP or GTP. Long-lasting depolarisations (>5 s) produced a progressive, reversible decay in the inward current despite the presence of intracellular ATP. Ca²⁺ channel blockade by Cd²⁺ unmasked a slowly activating outward Cs⁺ current flowing through a non-Ca²⁺ channel type, which became progressively unblocked by prolonged depolarisation even though Cs⁺ and TEA⁺ were present on both sides of the channel. The outward current waveform could be erroneously ascribed to a Ca- and/or voltage dependence of the Ca²⁺ macrocurrent. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

A patch-clamp study of the ionic selectivity of the large conductance,ca-activated chloride channel in muscle vesicles prepared fromAscaris suum

Summary Plasma membrane vesicles prepared from the bag re gion of the somatic muscle cell of the parasiteAscaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of anions has been investigated using the patch-clamp technique on isolated inside-out patches of muscle membrane. Symmetrical Cl solutions (140 mm) produced single-channel I/V plots with reversal potentials of 0 mV, substitution of bath Cl by 140 mM NO₃, Br and I caused depolarizing shifts in the reversal potentials. Replacement of the internal Cl by F (140 mM) caused a large hyperpolarizing shift in the reversal potential. The channel dis played a permeability sequence of I > Br = NO₃> Cl > F which differed from the corresponding conductance sequence Cl > NO₃ = Br = I > F. The ionic environment within the channel pore has been investigated using Reuter and Stevens (1980) plots to describe the selectivity and “fluidity” of the channel pore. In addition, the approach of Wright and Diamond (1977) was employed to estimate the number of cationic binding sites within the channel pore. The channel is relatively fluid but the number of cationic binding sites varies inversely with the ionic radius of the anion from 2.15 for F to 0.89 for the large planar anion NO₃     

Ca2+‐dependent activation of guard cell anion channels,triggered by hyperpolarization,is promoted by prolonged depolarization

Rapid stomatal closure is driven by the activation of S‐type anion channels in the plasma membrane of guard cells. This response has been linked to Ca²⁺ signalling, but the impact of transient Ca²⁺ signals on S‐type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca²⁺ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S‐type anion channels were monitored using intracellular triple‐barrelled micro‐electrodes. In cells kept at a holding potential of ?100 mV, voltage steps to ?180 mV triggered elevation of the cytosolic free Ca²⁺ concentration. The increase in the cytosolic Ca²⁺ level was accompanied by activation of S‐type anion channels. Guard cell anion channels were activated by Ca²⁺ with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of ?349 pA (SE = 107) at ?100 mV. Ca²⁺ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to ?100 mV, a transient increase in the cytosolic Ca²⁺ level was observed, activating S‐type channels without measurable delay. These data show that cytosolic Ca²⁺ elevation can activate S‐type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca²⁺ transport proteins, resulting in an overshoot of the cytosolic Ca²⁺ level after returning the membrane potential to ?100 mV.     

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al³⁺ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell.     

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger’ for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg²⁺ and/or H⁺ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP⁴⁻ in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.     

Volume-regulated anion channels serve as an auto/paracrine nucleotide release pathway in aortic endothelial cells

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.     

Association between SLCO1A2 genetic variation and methotrexate toxicity in human rheumatoid arthritis treatment

Methotrexate (MTX), one of the important disease‐modifying anti‐rheumatic drugs, is the first‐line drug for rheumatoid arthritis (RA) treatment. However, its adverse drug effects (ADEs) often lead to the abortion of MTX therapy. Human organic anion‐transporting polypeptide 1A2 (OATP1A2, also referred as OATP‐A or OATP1) encoded by SLCO1A2 gene is an important isoform of the solute carrier transporter (SLC) family. It is known to participate in the cellular uptake of MTX. In our previous study, we identified four OATP1A2 natural variants (E184K, D185N, T259P, and D288N) with impaired MTX uptake activity. This study aimed to evaluate the association of the SLCO1A2 genetic variations encoding these OATP1A2 variants and MTX‐related toxicity in RA patients. A total of 60 RA patients were genotyped for these four polymorphisms (G550A, G553A, A775C, and G862A). The association between SLCO1A2 genetic variations and MTX toxicity was analyzed by binary logistic regression analysis. Single nucleotide polymorphisms (SNPs) analysis revealed that A775C and G862A SNPs were not detected in RA patients enrolled in this study, and the presence of 550AA genotype was associated with a high risk of MTX ADEs. Haplotype analysis revealed that H3 (H3 = AG) showed a high risk of MTX ADEs. Furthermore, there was a significant association of 550AA genotype and impaired MTX disposition, which might be the cause of the increased incidence of MTX ADEs in RA patients. Therefore, genetic variations in SLCO1A2 gene are risk factors for MTX toxicity and its information contributes to the prediction of MTX‐related toxicity in RA treatment.     

Investigations on Antioxidant,Antiproliferative and COX‐2 Inhibitory Potential of Alkaloids from Anthocephalus cadamba (Roxb.) Miq. Leaves

In the present study, an ayurvedic medicinal plant, Anthocephalus cadamba (Roxb .) Miq . commonly known as ‘Kadamb’ was explored for its potential against oxidative stress and cancer. The fractions namely AC‐4 and ACALK (alkaloid rich fraction) were isolated from A. cadamba leaves by employing two different isolation methods and evaluated for their in vitro antioxidant and antiproliferative activity. The structure of the isolated AC‐4 was characterized tentatively as dihydrocadambine by using various spectroscopic techniques such as ESI‐QTOF‐MS, ¹H‐ and ¹³C‐NMR, DEPT, COSY, HMQC, and HMBC. Results of various antioxidant assays viz. 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), ABTS cation radical, superoxide anion radical scavenging, and plasmid nicking assay demonstrated that both the fractions viz. AC‐4 and ACALK possess ability to scavenge DPPH, ABTS radicals and effectively protected plasmid pBR322 DNA from damage caused by hydroxyl radicals. Further, when both fractions were evaluated for their potential to suppress growth of HeLa and COLO 205 cells, only ACALK fraction showed antiproliferative effects. ACALK exhibited GI₅₀ of 205.98 and 99.54 μg/ml in HeLa and COLO 205 cell lines, respectively. Results of Hoechst staining in cervical carcinoma (HeLa) cells confirmed that ACALK induced cell death in HeLa cells via apoptotic mode. Both the fractions also inhibited COX‐2 enzyme activity.     

Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.