Expression of Transient Receptor Potential Vanilloid Channels TRPV5 and TRPV6 in Human Blood Lymphocytes and Jurkat Leukemia T Cells

Regulation of Ca²⁺ entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca²⁺]_in; however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca²⁺-selective members of the TRPs, Ca²⁺ channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells. We found that TRPV6 and TRPV5 mRNAs are expressed in both Jurkat cells and quiescent HBLs; however, the levels of mRNAs were significantly higher in malignant cells than in quiescent lymphocytes. Western blot analysis showed TRPV5/V6 proteins in Jurkat T cells and TRPV5 protein in quiescent HBLs. However, the expression of TRPV6 protein was switched off in quiescent HBLs and turned on after mitogen stimulation of the cells with phytohemagglutinin. Inwardly directed monovalent currents that displayed characteristics of TRPV5/V6 currents were recorded in both Jurkat cells and normal HBLs. In outside–out patch-clamp studies, currents were reduced by ruthenium red, a nonspecific inhibitor of TRPV5/V6 channels. In addition, ruthenium red downregulated cell-cycle progression in both activated HBLs and Jurkat cells. Thus, we identified TRPV5 and TRPV6 calcium channels, which can be considered new candidates for Ca²⁺ entry into human lymphocytes. The correlation between expression of TRPV6 channels and the proliferative status of lymphocytes suggests that TRPV6 may be involved in the physiological and/or pathological proliferation of lymphocytes.     

Measurement of transmembrane pH gradients in human erythrocytes using 19F NMR

A new high-sensitivity method has been described for measuring transmembrane pH gradients in vesicular systems using ¹⁹F NMR. The ¹⁹F resonance of trifluoroethylamine has been shown to have a large pH-dependent chemical shift and the position of the resonance was measured with high precision and sensitivity. In suspensions of human erythrocytes, trifluoroethylamine distributed itself across the membrane and separate ¹⁹F resonances were obtained from the trifluoroethylamine inside and outside of the cells. The pH in each compartment was calculated from the resonance positions.     

The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate possible expression of Kv1.3 channels in other types of tissue, such as epithelia, binding experiments, immunoprecipitation studies and immunohistochemical studies were performed. The double-mutated, radiolabeled peptidyl ligand, ¹²⁵I-HgTX₁-A19Y/Y37F, which selectively binds Kv1.1, Kv1.2, Kv1.3 and Kv1.6 channels, was used to perform binding studies in epithelia isolated from rabbit kidney and colon. The equilibrium dissociation constant for this ligand was found to be in the sub-picomolar range and the maximal receptor concentration (in fmol/mg protein) 1.68 for colon and 0.61-0.75 for kidney epithelium. To determine the subtype of Kv1 channels, immunoprecipitation studies with ¹²⁵I-HgTX₁-A19Y/Y37F labeled epithelial membranes were performed with specific antibodies against Kv1.1, Kv1.2, Kv1.3, Kv1.4 or Kv1.6 subunits. These studies demonstrated that Kv1.3 subunits constituted more than 50% of the entire Kv1 subunit population. The precise localization of Kv1.3 subunits in epithelia was determined by immunohistochemical studies.     

Structural Studies of Bcl-xL/ligand Complexes using 19F NMR

Fluorine atoms are often incorporated into drug molecules as part of the lead optimization process in order to improve affinity or modify undesirable metabolic and pharmacokinetic profiles. From an NMR perspective, the abundance of fluorinated drug leads provides an exploitable niche for structural studies using ¹⁹F NMR in the drug discovery process. As ¹⁹F has no interfering background signal from biological sources, ¹⁹F NMR studies of fluorinated drugs bound to their protein receptors can yield easily interpretable and unambiguous structural constraints. ¹⁹F can also be selectively incorporated into proteins to obtain additional constraints for structural studies. Despite these advantages, ¹⁹F NMR has rarely been exploited for structural studies due to its broad lines in macromolecules and their ligand complexes, leading to weak signals in ¹H/¹⁹F heteronuclear NOE experiments. Here we demonstrate several different experimental strategies that use ¹⁹F NMR to obtain ligand–protein structural constraints for ligands bound to the anti-apoptotic protein Bcl-xL, a drug target for anti-cancer therapy. These examples indicate the applicability of these methods to typical structural problems encountered in the drug development process.     

Noninvasive NMR studies of metabolism in cultured Catharanthus roseus cells

Summary Nuclear magnetic resonance (NMR) spectroscopy provides a unique modality for the study of tissue-cultured plant cells. One of its major attractions is that it allows noninvasive studies of plant material. In addition, it can provide insight into the pH in the vacuole and cytoplasm, and into the compartmentalization of certain metabolites. In this review we show how phosphorus-31 NMR is used to study intracellular pH, phosphate uptake and storage, and energy metabolism in suspension cells of Catharanthus roseus. In addition, multinuclear NMR studies of the uptake of ammonium and the gradients of K⁺ over the membrane are discussed as well. The use of two-dimensional NMR for the study of whole cell extracts is also described. Finally, we show how nitrogen-14 and nitrogen-15 NMR are used to obtain information about the assimilation of inorganic sources in developing carrot somatic embryos. These NMR studies provide a unique insight into the metabolism of tissue-cultured plant cells.     

Monitoring of duplex and triplex formation by 19F NMR using oligodeoxynucleotides possessing 5-fluorodeoxyuridine unit as 19F signal transmitter

We prepared oligodeoxynucleotides (ODNs) possessing a 5-fluorodeoxyuridine (5-FU) unit as a ¹⁹F-signal transmitter, and characterized their structures including single strand, duplex, and triplex using ¹⁹F NMR. The change in chemical shift induced by incorporation of 5-FU into the ODNs and the formation of higher order structures allowed monitoring of structural changes. Data from UV melting experiments and CD spectra were consistent with the spectral changes in the NMR studies. These ¹⁹F-labeled ODNs may be promising molecular probes for the identification of DNA structures in complicated biological conditions.     

31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation

Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional³¹P and¹⁹F probes of intracellular pH (pH _c ) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na⁺ with choline or K⁺ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH _c on external Na⁺ concentration (c _Na ^o ). pH _c also depended on intracellular Na⁺ concentration (c _Na ^o ). Increasingc _Na ^c by withdrawing external K⁺ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc _Na ^o produced a larger acid shift in pH _c than with external K⁺ present. Comparison of separate preparations indicated that pH _c was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH _c of Swiss mouse 3T3 cells using³¹P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.     

Characterization of the human blood lymphocytes that produce a histamine-induced suppressor factor (HSF)

The cells which elaborate a soluble suppressor factor in vitro in response to histamine (histamine-induced suppressor factor or HSF) were partially characterized in the present studies. Human blood T- and B-cell populations were purified by affinity chromatography with rabbit anti-human F(Ab′)₂ and examined for their ability to make HSF. Highly purified populations of T cells, but not B cells, produced HSF in response to varying concentrations of histamine (10^?4 to 10^?4M). The HSF-producing cells were characterized further by means of affinity chromatography with columns containing conjugates of insolubilized histamine as well as by rosette formation with IgG (Tγ)- or IgM (Tμ)-coated ox red blood cells. These studies revealed the following: (a) Cells that synthesize HSF are retained on histamine (but not control) columns; (b) cells with histamine receptors comprise approximately 50% of the Tγ subpopulation but are not found in the Tμ subpopulation; (c) cells not retained by histamine columns have a reduced capacity to develop into suppressor cells following stimulation by concanavalin A or specific antigen (compared to unfractionated or control column passed cells). In addition, it was shown that cells synthesizing HSF predominantly express histamine type 2 receptors: (d)4-Methyl histamine (H₂ agonist), but not 2-methyl histamine (H₁ agonist), was capable of inducing HSF production; (e) cimetidine (H₂ antagonist) inhibited HSF production but chlorpheniramine (H₁ antagonist) did not. Taken together, these experiments suggest that T lymphocytes capable of expressing suppressor function following activation by histamine, specific antigen, concanavalin A, or perhaps through their Fc receptors may either be heterogeneous within the same subpopulation or more likely be the same cell with the complement of receptors described above.     

<Superscript>19</Superscript>F labelled glycosaminoglycan probes for solution NMR and non-linear (CARS) microscopy

Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of ¹⁹F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing ¹⁹F NMR spectroscopy is followed. Furthermore, the ability of ¹⁹F labelled GAGs to be imaged using CARS microscopy is demonstrated. ¹⁹F labelled GAGs enable both ¹⁹F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.     

Metabolism of glucose and glutamine in lymphocytes from graves' hyperthyroid patients: influence of methimazole treatment

Several studies have shown that thyroid hormones are able to influence selected immune responses such as cell mediated immunity, differentiation of B lymphocytes and the activity of NK cells. These hormones can also regulate the metabolism of glucose and glutamine in rat macrophages and their effects seem to occur mainly through the Krebs cycle. Alterations in the hexokinase, citrate synthase, glucose-6-phosphate dehydrogenase and glutaminase activities in lymphocytes from patients with Graves' disease, either untreated or on methimazole (MMI) therapy were investigated. Experiments were also done in vitro to determine the activities of these enzymes in normal lymphocytes cultured for 24 h in the presence of MMI, T₃ and T₄ using concentrations close to the physiological. Changes in the conversion of [U-¹⁴C]-glucose and [U-¹⁴C]-glutamine to ¹⁴CO₂ as caused by the addition of MMI, T₃ or T₄ to the culture medium were also evaluated. The results indicate that high levels of thyroid hormones might stimulate the metabolism of glucose and glutamine for a short period of time but, if the stimulus is maintained, the utilization of glutamine by lymphocytes is then suppressed. Moreover, MMI does affect lymphocyte metabolism but the significance of this finding for its immunosuppressive effect remains to be examined.     

Optimizing <Superscript>19</Superscript>F NMR protein spectroscopy by fractional biosynthetic labeling

In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, ¹⁹F and ¹H-¹⁵N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling. The effects of varying levels of enrichment of 3-fluorophenylalanine (i.e. between 3 and >95%) were further studied by ¹⁹F and ¹H-¹⁵N HSQC spectra,¹⁵N T₁ and T₂ relaxation measurements, ¹⁹F T₂ relaxation, translational diffusion and heat denaturation experiments via circular dichroism. Our results show that while several properties, including translational diffusion and thermal stability show little variation between non-fluorinated and >95% ¹⁹F labeled samples, ¹⁹F and ¹H-¹⁵N HSQC spectra show significant improvements in line widths and resolution at or below 76% enrichment. Moreover, high levels of fluorination (>80%) appear to increase protein disorder as evidenced by backbone ¹⁵N dynamics. In this study, reasonable signal to noise can be achieved between 60–76% ¹⁹F enrichment, without any detectable perturbations from labeling.     

HCO-3对植物生长发育及代谢的促进作用

重碳酸盐(bicarbonate, HCO^-₃)是碳酸盐岩经岩溶作用风化的产物,它深刻地影响着植物的生长发育和岩溶地区的生态环境。以往研究大都关注HCO^-₃对植物生长代谢的负面影响,如抑制植物的光合作用、降低碳氮代谢关键酶活性、破坏离子平衡等,少有人关注其对植物生长代谢的积极作用。该文依据前人的研究结果,综述了HCO^-₃对植物生长代谢的促进作用。已有的研究工作显示,HCO^-₃不仅在干旱等逆境胁迫下为植物提供短期的碳源和水源,促进气孔打开,恢复光合作用,而且通过调节碳氮代谢关键酶活性促进植物的碳氮代谢,参与调控植物的碳同化和氮还原等复杂的生理过程; 此外,HCO^-₃还通过影响葡萄糖代谢歧化,改变植物糖酵解途径和磷酸戊糖途径的分配,以增强植物的抗逆能力,从而获取生存机会。HCO^-₃的这些积极作用不仅使之成为促进植物生理代谢的关键因子,而且成为连接光合作用和岩溶作用的纽带。阐明HCO^-₃对植物生长发育的积极作用,可为维护喀斯特生态系统的生物多样性和稳定性、优化喀斯特生态系统功能提供理论依据。     

Role of the Na+/H+ antiport in the regulation of the internal pH of Ehrlich ascites tumor cells in culture

Summary Ehrlich ascites tumor cells contain a Na⁺ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC₅₀ of 25 m and by dimethylamiloride with an IC₅₀ of 0.6 m at 1mm external Na⁺. Decrease of external Na⁺ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na⁺/H⁺ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na⁺/H⁺ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na⁺ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na⁺. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na⁺ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na⁺/H⁺ antiporter to internal protons. The Na⁺/H⁺ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.     

Epithelial V-Like Antigen Mediates Efficacy of Anti-Alpha4 Integrin Treatment in a Mouse Model of Multiple Sclerosis

Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha₄ integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA⁺ lymphocytes following immunization. Following active induction of EAE using the MOG^35–55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19⁺, CD21⁺, sIgG⁺), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5⁺IgG⁺ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19⁺, CD21⁺, sIgG⁺ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19⁺, CD21⁺, sIgG⁺ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders.     

The effect of the cosolvent trifluoroethanol on a tryptophan side chain orientation in the hydrophobic core of troponin C

The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N‐domain of cardiac troponin C (F77W‐cNTnC) to study the domain orientation of troponin C in muscle fibers using solid‐state NMR. In our previous study, we determined the NMR structure of calcium‐saturated mutant F77W‐V82A‐cNTnC in the presence of 19% 2,2,2‐trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W‐cNTnC in presence of its biological binding partner troponin I peptide (cTnI_144–163) and in the absence of TFE. We have also characterized a panel of six F77W‐cNTnC structures in the presence and absence TFE, cTnI_144–163, and the extra mutation V82A, and used ¹⁹F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents.     

α-Trifluoromethyl-β-Ureido-Propionic Acid (F3MUPA): A New Metabolite of Trifluridine (F3TdR)

Abstract

The metabolism of 5-trifluro-2′-deoxythymidine (trifluridine; F₃TdR) in male BALB/c mice bearing EMT-6 tumors has been investigated using ¹9F NMR spectroscopy. We previously (Tandon et al, 1992) reported the detection and identification of 5,6-dihydro-5-trifluorothymine (DHF₃T, 3) and 5,6-dihydroxy-5-trifluorothymine (DOHF₃T) as new metabolites of F₃TdR in mice urine. Further exploration of the metabolism of trifluridine has led to the identification of α-trifluoromethyl-β-ureido-propionic acid (F₃MUPA, 4) as a previously unreported metabolite in the urine of F₃TdR treated mice. Authentic F₃MUPA was obtained by synthesis via an established route. A comparison of chemical shift and the H-F coupling constant of an authentic sample, with the ¹⁹F signal from urine, indicated the presence of F₃MUPA in murine urine. Mixing crude urine with authentic F₃MUPA resulted in the enhancement of the corresponding fluorine signal without affecting or introducing others, thereby confirming the presence of F₃MUPA as a urinary metabolite.     

Sister-chromatid exchange in highly purified human CD4 and CD8 lymphocytes

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNA^Met_f has been further characterized. This substituent is responsible for the ¹⁹F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution ¹⁹F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield ¹⁹F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNA^Met_m and tRNA^Val_l, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with ¹⁹F spectrum of the model compound 5′-deoxy-5-fluoro-5,6-dihydrouridine (dH⁵₆FUrd) (δ_FUra = ? 31.4 ppm; J_HF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH⁵₆FUrd is considerably more alkali labile than 5,6-dihydrouridine (H⁵₆Urd). At pH 8.9, where H⁵₆Urd is stable, dH⁵₆FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a ¹⁹F resonance at ? 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The ¹⁹F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the ¹⁹F signal at ? 15 ppm. Hydrolysis of this tRNA with RNAase T₂ produces a sharp doublet 33 ppm upfield (J_HF = 45 Hz). Similarities of the ¹⁹F chemical shift and coupling constant to those of dH⁵₆FUrd, allows assignment of the peak at ? 33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.     

Difluorophosphate as a 19F NMR probe of erythrocyte membrane potential

Erythrocyte membrane potential can be estimated by measuring the transmembrane concentration (activity) distribution of a membrane-permeable ion. We present here the study of difluorophosphate (DFP) as a ¹⁹F NMR probe of membrane potential. This bicarbonate and phosphate analogue has a pK_a of 3.7±0.2 (SD, n = 4) and therefore exists almost entirely as a monovalent anion at physiological pH. When it is incorporated into red cell suspensions, it gives two well resolved resonances that arise from the intra- and extracellular populations; the intracellular resonance is shifted 130 Hz to higher frequency from that of the extracellular resonance. Hence the transmembrane distribution of DFP is readily assessed from a single ¹⁹F NMR spectrum and the membrane potential can be calculated using the Nernst equation. The membrane potential was independent of, DFP concentration in the range 4 to 59 mM, and haematocrit of the cell suspensions of 31.0 to 61.4%. The membrane potential determined by using DFP was 0.94±0.26 of that estimated from the transmembrane pH difference. The distribution ratios of intracellular/extracellular DFP were similar to those of the membrane potential probes, hypophosphite and trifluoroacetate. DFP was found to be transported across the membranes predominantly via the electrically-silent pathway mediated by capnophorin. Using magnetization transfer techniques, the membrane influx permeability-coefficient of cells suspended in physiological medium was determined to be 7.2±2.5 × 10^–6 cm s^–1 (SD, n=4). Offprint requests to: P. W Kuchel