The antimalarial artemisinin is a non-electrophilic agonist of the transient receptor potential ankyrin type 1 receptor-channel.

Artemisinin and its derivatives are the main therapeutic drugs against Plasmodium protists, the causative agents of malaria. While several putative mechanisms of action have been proposed, the precise molecular targets of these compounds have not been fully elucidated. In addition to their antimalarial properties, artemisinins have been reported to act as anti-tumour agents and certain antinociceptive effects have also been proposed. We investigated the effect of the parent compound, artemisinin, on a number of temperature-gated Transient Receptor Potential ion channels (so called thermoTRPs), given their demonstrated roles in pain-sensing and cancer. We report that artemisinin acts as an agonist of the Transient Receptor Potential Ankyrin type 1 (TRPA1) receptor channel. Artemisinin was able to evoke calcium transients in HEK293T cells expressing recombinant human TRPA1, as well as in a subpopulation of mouse dorsal root ganglion (DRG) neurons which also responded to the selective TRPA1 agonist allyl isothiocyanate (AITC) and these responses were reversibly abolished by the selective TRPA1 antagonist A967079. Artemisinin also triggered whole-cell currents in HEK293T cells transiently transfected with human TRPA1, as well as in TRPA1-expressing DRG neurons, and these currents were inhibited by A967079. Interestingly, using human TRPA1 mutants, we demonstrate that artemisinin acts as a non-electrophilic agonist of TRPA1, activating the channel in a similar manner to carvacrol and menthol. These results may provide a better understanding of the biological actions of the very important antimalarial and anti-tumour agent artemisinin.

Lithium rescues dendritic abnormalities in Ank3 deficiency models through the synergic effects of GSK3ß and cyclic AMP signaling pathways.

Bipolar disorder (BD) is a highly heritable mood disorder with intermittent episodes of mania and depression. Lithium is the first-in-line medication to treat BD, but it is only effective in a subset of individuals. Large-scale human genomic studies have repeatedly linked the ANK3 gene (encoding ankyrin-G, AnkG) to BD. Ank3 knockout mouse models mimic BD behavioral features and respond positively to lithium treatment. We investigated cellular phenotypes associated with BD, including dendritic arborization of pyramidal neurons and spine morphology in two models: (1) a conditional knockout mouse model which disrupts Ank3 expression in adult forebrain pyramidal neurons, and (2) an AnkG knockdown model in cortical neuron cultures. We observed a decrease in dendrite complexity and a reduction of dendritic spine number in both models, reminiscent of reports in BD. We showed that lithium treatment corrected dendrite and spine deficits in vitro and in vivo. We targeted two signaling pathways known to be affected by lithium using a highly selective GSK3ß inhibitor (CHIR99021) and an adenylate cyclase activator (forskolin). In our cortical neuron culture model, CHIR99021 rescues the spine morphology defects caused by AnkG knockdown, whereas forskolin rescued the dendrite complexity deficit. Interestingly, a synergistic action of both drugs was required to rescue dendrite and spine density defects in AnkG knockdown neurons. Altogether, our results suggest that dendritic abnormalities observed in loss of function ANK3 variants and BD patients may be rescued by lithium treatment. Additionally, drugs selectively targeting GSK3ß and cAMP pathways could be beneficial in BD.

Effect of Mercury on Membrane Proteins, Anionic Transport and Cell Morphology in Human Erythrocytes.

BACKGROUND/AIMS: Mercury (Hg) is a heavy metal widespread in all environmental compartments as one of the most hazardous pollutants. Human exposure to this natural element is detrimental for several cellular types including erythrocytes (RBC) that accumulate Hg mainly bound to the SH groups of different cellular components, including protein cysteine residues. The cellular membrane represents a major target of Hg-induced damage in RBC with loss of physiological phospholipid asymmetry, due to phosphatidylserine (PS) exposure to the external membrane leaflet. To investigate Hg-induced cytotoxicity at the molecular level, the possible interaction of this heavy metal with RBC membrane proteins was investigated. Furthermore, Hg-induced alterations in band 3 protein (B3p) transport function, PS-exposing macrovesicle (MVs) formation and morphological changes were assessed. METHODS: For this aim, human RBC were treated in vitro with different HgCl2 concentrations (range 10-40 µM) and the electrophoretic profile of membrane proteins as well as the expression levels of Ankyrin and Flottilin-2 evaluated by SDS-PAGE and Western blot, respectively. The effect of alterations in these proteins on RBC morphology was evaluated by digital holographic microscopy and anionic transport efficiency of B3p was evaluated as sulphate uptake. Finally, PS- bearing MVs were quantified by annexin-V binding using FACS analysis. RESULTS: Findings presented in this paper indicate that RBC exposure to HgCl2 induces modifications in the electrophoretic profile of membrane protein fraction. Furthermore, our study reveals the Hg induced alterations of specific membrane proteins, such as Ankyrin, a protein essential for membrane-cytoskeleton linkage and Flotillin-2, a major integral protein of RBC lipid rafts, likely responsible for decreased membrane stability and increased fragmentations. Accordingly, under the same experimental conditions, RBC morphological changes and PS-bearing MVs release are observed. Finally, RBC treatment significantly affects the B3p-mediated anionic transport, that we report reduced upon HgCl2 treatment in a dose dependent manner. CONCLUSION: Altogether, the findings reported in this paper confirm that RBC are particularly vulnerable to Hg toxic effect and provide new insight in the Hg-induced protein modification in human RBC affecting the complex biological system of cellular membrane. In particular, Hg could induce dismantle of vertical cohesion between the plasma membrane and cytoskeleton as well as destabilization of lateral linkages of functional domains. Consequently, decreased membrane deformability could impair RBC capacity to deal with the shear forces in the circulation increasing membrane fragmentations. Furthermore, findings described in this paper have also significant implication in RBC physiology, particularly related to gas exchanges.

Beyond antibodies: ankyrins and DARPins. From basic research to drug approval.

This Pharmacological Perspective describes the pathway that, starting from the deep understanding of ankyrins - a family of proteins with high variability-binding and high specificity-binding characteristics - led to the development of a new class of recombinant-binding proteins, the DARPins (designed ankyrin repeat proteins). These are envisaged as alternatives to mAbs and related biologics, with the potential to overcome certain shortcomings of mAbs. DARPins have relatively low molecular weights (14-21kDas) and more favorable PK profiles than mAbs, are stable proteins that can be easily produced in Escherichia coli and can be used in their monovalent form or conjugated to other moieties, for example, polyethylene glycol (PEG) to enhance their half-life. DARPins can also be engineered to produce bi-specific or tri-specific compounds that bind different epitopes of the same target or two different targets. Abicipar, a first-in-class anti-VEGF-A DARPin had similar efficacy compared to anti-VEGF biologics (bevacizumab, ranibizumab) in preclinical studies and was not inferior to ranibizumab in the treatment of age-related macular degeneration (AMD) with a reduced number of intravitreal injections. Abicipar has recently been submitted for regulatory approval for use in AMD.

Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1.

BACKGROUND: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity. OBJECTIVE: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs. METHOD: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI). RESULTS: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity. CONCLUSIONS: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of AnkGAG1D4 ankyrin for its CA target. AnkGAG1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future.

A virulence factor encoded by a polydnavirus confers tolerance to transgenic tobacco plants against lepidopteran larvae, by impairing nutrient absorption.

The biological control of insect pests is based on the use of natural enemies. However, the growing information on the molecular mechanisms underpinning the interactions between insects and their natural antagonists can be exploited to develop "bio-inspired" pest control strategies, mimicking suppression mechanisms shaped by long co-evolutionary processes. Here we focus on a virulence factor encoded by the polydnavirus associated with the braconid wasp Toxoneuron nigriceps (TnBV), an endophagous parasitoid of noctuid moth larvae. This virulence factor (TnBVANK1) is a member of the viral ankyrin (ANK) protein family, and appears to be involved both in immunosuppression and endocrine alterations of the host. Transgenic tobacco plants expressing TnBVANK1 showed insecticide activity and caused developmental delay in Spodoptera littoralis larvae feeding on them. This effect was more evident in a transgenic line showing a higher number of transcripts of the viral gene. However, this effect was not associated with evidence of translocation into the haemocoel of the entire protein, where the receptors of TnBVANK1 are putatively located. Indeed, immunolocalization experiments evidenced the accumulation of this viral protein in the midgut, where it formed a thick layer coating the brush border of epithelial cells. In vitro transport experiments demonstrated that the presence of recombinant TnBVANK1 exerted a dose-dependent negative impact on amino acid transport. These results open new perspectives for insect control and stimulate additional research efforts to pursue the development of novel bioinsecticides, encoded by parasitoid-derived genes. However, future work will have to carefully evaluate any effect that these molecules may have on beneficial insects and on non-target organisms.

Crystal structure of an antiviral ankyrin targeting the HIV-1 capsid and molecular modeling of the ankyrin-capsid complex.

Ankyrins are cellular repeat proteins, which can be genetically modified to randomize amino-acid residues located at defined positions in each repeat unit, and thus create a potential binding surface adaptable to macromolecular ligands. From a phage-display library of artificial ankyrins, we have isolated Ank(GAG)1D4, a trimodular ankyrin which binds to the HIV-1 capsid protein N-terminal domain (NTD(CA)) and has an antiviral effect at the late steps of the virus life cycle. In this study, the determinants of the Ank(GAG)1D4-NTD(CA) interaction were analyzed using peptide scanning in competition ELISA, capsid mutagenesis, ankyrin crystallography and molecular modeling. We determined the Ank(GAG)1D4 structure at 2.2 Å resolution, and used the crystal structure in molecular docking with a homology model of HIV-1 capsid. Our results indicated that NTD(CA) alpha-helices H1 and H7 could mediate the formation of the capsid-Ank(GAG)1D4 binary complex, but the interaction involving H7 was predicted to be more stable than with H1. Arginine-18 (R18) in H1, and R132 and R143 in H7 were found to be the key players of the Ank(GAG)1D4-NTD(CA) interaction. This was confirmed by R-to-A mutagenesis of NTD(CA), and by sequence analysis of trimodular ankyrins negative for capsid binding. In Ank(GAG)1D4, major interactors common to H1 and H7 were found to be S45, Y56, R89, K122 and K123. Collectively, our ankyrin-capsid binding analysis implied a significant degree of flexibility within the NTD(CA) domain of the HIV-1 capsid protein, and provided some clues for the design of new antivirals targeting the capsid protein and viral assembly.

Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4-induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1.

The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B4 (LTB4) that is up-regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle-injected (0.1% bovine serum albumin-saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC-030031 (100 mg/kg; 76%). Leukotriene B4 receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1-knockout (TRPV1-KO) nor TRPA1-knockout (TRPA1-KO mice exhibited LTB4-induced itch compared with their wild-type counterparts. The reactive oxygen species scavengers N-acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4-induced superoxide release was attenuated by TCS 5861528 (56%) and HC-030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U-75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1-KO mice showed impaired myeloperoxidase release, whereas TRPA1-KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein.

BACKGROUND: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. RESULTS: A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 µM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway. CONCLUSIONS: The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.

The Na(+)/Ca(2+)-exchanger: an essential component in the mechanism governing cardiac steroid-induced slow Ca(2+) oscillations.

Plasma membrane (PM) Na(+), K(+)-ATPase, plays crucial roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically bind to the Na(+), K(+)-ATPase and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds, synthesized in and released from the adrenal gland, are considered a new family of steroid hormones. Previous studies showed that ouabain induces slow Ca(2+) oscillations in COS-7 cells by enhancing the interactions between Na(+), K(+)-ATPase, inositol 1,4,5-trisphosphate receptor (IP(3)R) and Ankyrin B (Ank-B) to form a Ca(2+) signaling micro-domain. The activation of this micro-domain, however, is independent of InsP3 generation. Thus, the mechanism underlying the induction of these slow Ca(2+) oscillations remained largely unclear. We now show that other CS, such as bufalin, can also induce Ca(2+) oscillations. These oscillations depend on extracellular Ca(2+) concentrations [Ca(2+)](out) and are inhibited by Ni(2+). Furthermore, we found that these slow oscillations are Na(+)(out) dependent, abolished by Na(+)/Ca(2+) exchanger1 (NCX1)-specific inhibitors and markedly attenuated by NCX1 siRNA knockdown. Based on these results, a model is presented for the CS-induced slow Ca(2+) oscillations in COS-7 cells.

TRPA1 and sympathetic activation contribute to increased risk of triggered cardiac arrhythmias in hypertensive rats exposed to diesel exhaust.

BACKGROUND: Diesel exhaust (DE), which is emitted from on- and off-road sources, is a complex mixture of toxic gaseous and particulate components that leads to triggered adverse cardiovascular effects such as arrhythmias. OBJECTIVE: We hypothesized that increased risk of triggered arrhythmias 1 day after DE exposure is mediated by airway sensory nerves bearing transient receptor potential (TRP) channels [e.g., transient receptor potential cation channel, member A1 (TRPA1)] that, when activated by noxious chemicals, can cause a centrally mediated autonomic imbalance and heightened risk of arrhythmia. METHODS: Spontaneously hypertensive rats implanted with radiotelemeters were whole-body exposed to either 500 µg/m³ (high) or 150 µg/m³ (low) whole DE (wDE) or filtered DE (fDE), or to filtered air (controls), for 4 hr. Arrhythmogenesis was assessed 24 hr later by continuous intravenous infusion of aconitine, an arrhythmogenic drug, while heart rate (HR) and electrocardiogram (ECG) were monitored. RESULTS: Rats exposed to wDE or fDE had slightly higher HRs and increased low-frequency:high-frequency ratios (sympathetic modulation) than did controls; ECG showed prolonged ventricular depolarization and shortened repolarization periods. Rats exposed to wDE developed arrhythmia at lower doses of aconitine than did controls; the dose was even lower in rats exposed to fDE. Pretreatment of low wDE-exposed rats with a TRPA1 antagonist or sympathetic blockade prevented the heightened sensitivity to arrhythmia. CONCLUSIONS: These findings suggest that a single exposure to DE increases the sensitivity of the heart to triggered arrhythmias. The gaseous components appear to play an important role in the proarrhythmic response, which may be mediated by activation of TRPA1, and subsequent sympathetic modulation. As such, toxic inhalants may partly exhibit their toxicity by lowering the threshold for secondary triggers, complicating assessment of their risk.

CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics.

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.

DARPins: a new generation of protein therapeutics.

DARPins (designed ankyrin repeat proteins) are a novel class of binding molecules with the potential to overcome limitations of monoclonal antibodies, hence allowing novel therapeutic approaches. DARPins are small, single domain proteins (14 kDa) which can be selected to bind any given target protein with high affinity and specificity. These characteristics make them ideal agonistic, antagonistic or inhibitory drug candidates. Furthermore, DARPins can be engineered to carry various effector functions or combine multiple binding specificities, enabling completely new drug formats. Taken together, DARPins are a prominent member of the next generation of protein therapeutics with the potential to surpass existing antibody drugs.

Functional significance of axonal Kv7 channels in hippocampal pyramidal neurons.

Members of the Kv7 family (Kv7.2-Kv7.5) generate a subthreshold K(+) current, the M- current. This regulates the excitability of many peripheral and central neurons. Recent evidence shows that Kv7.2 and Kv7.3 subunits are targeted to the axon initial segment of hippocampal neurons by association with ankyrin G. Further, spontaneous mutations in these subunits that impair axonal targeting cause human neonatal epilepsy. However, the precise functional significance of their axonal location is unknown. Using electrophysiological techniques together with a peptide that selectively disrupts axonal Kv7 targeting (ankyrin G-binding peptide, or ABP) and other pharmacological tools, we show that axonal Kv7 channels are critically and uniquely required for determining the inherent spontaneous firing of hippocampal CA1 pyramids, independently of alterations in synaptic activity. This action was primarily because of modulation of action potential threshold and resting membrane potential (RMP), amplified by control of intrinsic axosomatic membrane properties. Computer simulations verified these data when the axonal Kv7 density was three to five times that at the soma. The increased firing caused by axosomatic Kv7 channel block backpropagated into distal dendrites affecting their activity, despite these structures having fewer functional Kv7 channels. These results indicate that axonal Kv7 channels, by controlling axonal RMP and action potential threshold, are fundamental for regulating the inherent firing properties of CA1 hippocampal neurons.

DARPins: a true alternative to antibodies.

Designed ankyrin repeat proteins (DARPins) are a promising class of non-immunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and drug development. DARPins have been successfully used, for example, for the inhibition of kinases, proteases and drug-exporting membrane proteins. DARPins specifically targeting the cancer marker HER2 have also been generated and were shown to function in both in vitro diagnostics and in vivo tumor targeting. DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads because of their favorable molecular properties, including small size and high stability. The low-cost production in bacteria and the rapid generation of many target-specific DARPins make the DARPin approach useful for drug discovery. Additionally, DARPins can be easily generated in multispecific formats, offering the potential to target an effector DARPin to a specific organ or to target multiple receptors with one molecule composed of several DARPins.

Evidence for an interaction between adducin and Na(+)-K(+)-ATPase: relation to genetic hypertension.

Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

Decreased content of protein 4.2 in ankyrin-deficient normoblastosis (nb/nb) mouse red blood cells: evidence for ankyrin enhancement of protein 4.2 membrane binding.

Homozygous normoblastosis (nb/nb) mice, whose red blood cell (RBC) membranes are nearly completely deficient in full-length 210-kD ankyrin, were used to study interactions between ankyrin and protein 4.2 (P4.2). Although it is unclear whether or not these proteins interact in the membrane, both ankyrin and P4.2 bind to the cytoplasmic domain of band 3 (cdb3). In addition to the complete deficiency of full-length ankyrin, nb/nb RBC membranes are also partially spectrin deficient, resulting in morphologically spherocytic and mechanically fragile cells. A new finding was that nb/nb RBC membranes are severely (approximately 73%) P4.2 deficient compared with wild type (+/+) or high reticulocyte mouse RBC membranes. Metabolic labeling of nb/nb reticulocytes showed active P4.2 synthesis at levels comparable with high reticulocyte controls suggesting that the nb/nb P4.2 deficiency was not the result of defective P4.2 synthesis. Reconstitution of nb/nb inside-out vesicles (IOVs) with human RBC ankyrin restored ankyrin levels to approximately 80% of +/+ IOV levels and increased binding of exogenously added human RBC P4.2 by approximately 60%. These results suggest that ankyrin is required for normal associations of P4.2 with the RBC membrane.

Ankyrin inhibits binding of erythrocyte spectrin to phospholipid vesicles.

The studies on binding of erythrocyte spectrin to frozen and thawed phospholipid liposomes and its inhibition by ankyrin were performed. It was found that ankyrin inhibited up to 60% binding of spectrin by phosphatidylethanolamine/phosphatidylcholine vesicles. It was able to dissociate up to 40% of spectrin from this complex. Ankyrin inhibition of binding of phosphatidylserine/phosphatidylcholine vesicles by spectrin, although much lower, was also observed.