Novel splice variants of the voltage-sensitive sodium channel alpha subunit

In this study, we have identified novel splice variants of the Na+ channel alpha subunit mRNA from cultured rat astrocytes and neuroblastoma cells. These splice variants are characterized by premature truncation or deletion of a segment in the third domain of the Na+ channel alpha subunit. The expression of three splice variants was upregulated by exposure to 1 mM dibutyryl cAMP in spinal cord astrocytes but not in cerebral astrocytes and in B50 and B104 neuroblastoma cells. The calcium ionophore 1 microM A23187, did not influence the expression of splice variants in either astrocytes or neuroblastoma cells. These findings suggest that spinal cord astrocytes may maintain a unique regulatory pathway that participates in the control of Na+ channel mRNA expression.     

Complex association and dissociation kinetics of brevetoxin binding to voltage-sensitive rat brain sodium channels

31P magnetic resonance spectroscopy measurements of pH and the concentrations of orthophosphate and phosphocreatine were used to estimate rates of glycogenolytic and oxidative ATP synthesis in rat leg muscle during 6 min sciatic nerve stimulation at different rates (1-4 Hz). To study the regulation of glycogenolysis during exercise, the apparent 'glycogenolytic capacity' (L(MAX)) was calculated from glycogenolytic ATP synthesis rate and orthophosphate concentration as a measure of the Ca2+-dependent activation of glycogen phosphorylase. This was found to be proportional to the total ATP synthesis rate (F), and to decline with time; expressed relative to total ATP turnover rate as L(MAX)/F, its initial value was 2.9+/-0.6, declining with half-time 1.4+/-0.4 min. The apparent 'mitochondrial capacity' (Q(MAX)), calculated from oxidative ATP synthesis rate and [ADP], was independent of ATP turnover rate, but increased with half-time 0.8+/-0.1 min to 29+/-2 mmol kg(-1) min(-1): thus [ADP] was the predominant but not the only influence on oxidative ATP synthesis. Numerical simulation shows that time-dependent changes in L(MAX)/F exert a strong influence on pH and on the concentrations of phosphocreatine and ADP.     

Differences in the binding sites of two site-3 sodium channel toxins

Site-3 toxins from scorpion and sea anemone bind to Na channels and selectively inhibit current decay. Anthopleurins A and B (ApA and ApB, respectively), toxins found in the venom of the sea anemone Anthopleura xanthogrammica, bind to closed states of mammalian skeletal and cardiac Na channels with differing affinities which arise from differences in first-order toxin/channel dissociation rate constants, koff. Using chimera comprising domain interchanges between channel isoforms, we examined the structural basis of this differential affinity. Toxin/channel association rates, kon, were similar for both toxins and both parental channels. Domain 4 determined koff for ApA, while ApB dissociated from all tested chimera in a cardiac-like manner. To probe this surprising difference between two such closely related toxins, we examined the interaction of chimeric channels with a form of ApB in which the two nonconserved basic residues, Arg-12 and Lys-49, were converted to the corresponding neutral amino acids from ApA. In the chimera comprising domain 1 from the cardiac muscle isoform and domains 2-4 from the skeletal muscle isoform, toxin dissociated at a rate intermediate between those of the parental channels. We conclude that the differential component of ApA binding is controlled by domain 4 and that some component of ApB binding is not shared by ApA. This additional component probably binds to an interface between channel domains and is partly mediated by toxin residues Arg-12 and Lys-49.     

Permeability of the sodium channel in Myxicola to organic cations

Honeybee venom was separated into seven fractions by gel filtration on Sephadex G-75. Allergenic activities of these fractions were assessed by the paper disc radioallergosorbent test (RAST) with a panel of sera from 24 individuals who had systemic reactions to bee stings, 7 who had large local reactions, and 10 control subjects who had reactions of 5 cm or less following bee stings. Three fractions were identified by enzyme or direct hemolytic activity. Twenty-nine of 31 sera from patients having either systemic or large local reactions to bee stings were positive when radioallergosorbent tested with whole bee venom; 22 were positive to phospholipase A, and 28 were positive to both fractions 1 and 2. Thirteen sera combined most strongly with fraction 1, 12 sera most strongly with fraction 2, hyaluronidase, and three sera about equally with fractions 1, 2, and 3. Reactions with other fractions were much weaker. Fractions 1 and 2 were potent inhibitors of RAST with whole venom in the sera reacting most strongly with fractions 1 or 2, respectively. Fraction 3, phospholipase A, and commercial bee venom phospholipase A were significantly less potent inhibitors with the sera tested. In the cases in which IgE antibody bindings to fractions 1, 2, and 3 were of similar magnitude, inhibitions of RAST using the various fractions both on the discs and as inhibitors demonstrated substantial cross-reactivity between the fractions. These results strongly indicate that by using RAST with human sera from bee sting-sensitive individuals, fraction 1, the high molecular materials, and fraction 2, hyaluronidase, are the major allergens in honeybee venom. Phospholipase A appears to be of secondary importance.     

3-Phosphohistidine cannot replace phosphotyrosine in high-affinity binding to phosphotyrosine binding or Src homology 2 domains

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Phosphorylated tyrosine residues can serve as association sites for other proteins in signal transduction cascades of tyrosine kinase receptors. Formation of phosphohistidine residues in proteins has been found in eukaryotic and prokaryotic organisms. Furthermore, it has been suggested that phosphohistidine might substitute for phosphotyrosine in conferring high-affinity binding to proteins involved in signal transduction. We have analyzed the ability of 3-phosphohistidine to associate with the known phosphotyrosine-specific phosphotyrosine binding and src homology 2 protein domains. From our binding studies using synthetic peptides, we conclude that 3-phosphohistidine cannot replace phosphotyrosine in conferring high-affinity binding to the phosphotyrosine binding domain of shc or the src homology 2 domain of phospholipase C-gamma1.     

The cytoplasmic domain of bovine herpesvirus 1 glycoprotein B is important for maintaining conformation and the high-affinity binding site of gB

Bovine herpesvirus 1 (BHV-1) glycoprotein B (gB) has been shown to interact with two types of receptor on Madin Darby bovine kidney cells. The first receptor is heparan sulfate proteoglycan, whereas the second high-affinity receptor remains unknown. In order to study the structural requirement for gB's high-affinity binding activity, different forms of the gB ectodomain were expressed and compared with authentic gB. By using chemical cross-linking and sucrose gradient centrifugation, we found that BHV-1 gB was able to form dimers. A region between the cleavage site and the transmembrane anchor region, residues 506 to 763, was found to be required for gB oligomerization. Although the purified gBt and gBtM, two truncated forms of gB, formed oligomers, they did not block the high-affinity cellular receptor, suggesting that oligomerization was not the reason for the loss of the high-affinity binding site on gB. However, an N-terminal juxtamembrane region-located epitope recognized by a monoclonal antibody, designated epitope I, was lost from gBt and gBtM, indicating that both truncated gBs are conformationally changed. Therefore, the structure around this particular region may be required for the existence of the gB high-affinity binding site.     

Estrogen receptor alpha requires no accessory factors for high-affinity binding to a consensus response element

Estrogen receptor (ER) alpha is commonly thought to bind to a consensus estrogen response element (ERE) as a homodimer, but previous experiments have not ruled out the presence of other proteins in the ERalpha/ERE complex. To characterize this interaction in more detail, we overexpressed mouse (m) ERalpha in a baculovirus system, using the selective advantage of the apoptosis inhibitor p35. Recombinant mERalpha possesses the predicted molecular weight and binds 17beta-estradiol and an oligonucleotide containing a consensus vitellogenin ERE with high affinity. Over a wide concentration range of mERalpha protein (0.1-50 nM), only one complex was detected between mERalpha and vitellogenin ERE in gel shift assays. The ratio of E2:vitellogenin ERE bound by mERalpha was close to 2:1, and each complex contained only one ERE. The molecular weight of the complex was determined to be 160 000, very close to that predicted for two mERalpha proteins and one ERE oligonucleotide, therefore providing strong evidence that no other proteins were present. Recombinant mERalpha was purified such that it was the only protein observable by silver stain. Purified mERalpha and mERalpha in a nuclear extract behaved identically in Ferguson analysis, providing more evidence that only mERalpha was binding to the ERE. Purified mERalpha bound vitellogenin ERE with high affinity (Kd = 0. 92 +/- 0.20 nM), indicating that no other proteins are necessary for high-affinity mERalpha interaction with a consensus ERE. To determine whether ERalpha in an estrogen-responsive mammalian tissue behaves the same as the overexpressed mERalpha, we tested rat uterine cytosol by Ferguson analysis. ERalpha in rat uterine cytosol behaved identically to overexpressed mERalpha, suggesting that ERalpha in the uterine extract also binds to DNA predominantly as a homodimer with no additional proteins.     

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.     

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

BACKGROUND: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. RESULTS: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. CONCLUSIONS: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.     

Halogenated chalcones with high-affinity binding to P-glycoprotein: potential modulators of multidrug resistance

Previous studies have shown that flavonoids are modulators of the transmembrane P-glycoprotein (P-gp) which mediates cell multidrug resistance. Some structural elements have been identified which seem to contribute to these compounds' activity. In the present study, a series of halogenated chalcones was prepared to further explore the structural requirements for the P-gp modulation. Four halogenated chalcones have been synthesized and evaluated as potential modulators of P-gp-mediated multidrug resistance of cancer cells by in vitro assays using a purified recombinant domain of the transporter containing the modulator binding site. Halogenated chalcones exhibited high-affinity binding, the 2',4', 6'-trihydroxy-4-iodochalcone behaving as the most potent compound with a KD value in the nanomolar range.     

Identification of a new high-affinity binding protein for neurotoxic phospholipases A2

We have examined the relationship between the procoagulant activity of F3II mouse mammary carcinoma cells and the production of urokinase, a profibrinolytic serine protease involved in tumor invasion and hematogenous metastasis. F3II cells were capable of inducing the conversion of purified fibrinogen to fibrin in the presence of calcium and plasma traces. Immunocytochemical examination of semi-confluent monolayers demonstrated that F3II cells also synthesized high levels of urokinase. Although fibrinogen did not modify profibrinolytic activity produced by F3II monolayers, fibrin formation increased tumor-derived urokinase activity by two-fold. The present data provide new insights into the cooperative role of coagulation and fibrinolysis facilitating and perpetuating tumor invasion.     

Subunit stoichiometry of the epithelial sodium channel

The epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed alpha, beta, and gamma. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an alpha2 beta gamma stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

Identification of binding sites for calcium channel antagonists

The binding sites of three typical calcium channel antagonists, 1, 4-dihydropyridines, benzothiazepines and phenylalkylamines, were successfully identified within the primary structures of calcium channels using a photoaffinity labeling technique. The results confirm pharmacological observations of the three antagonists that had been proposed to interact allosterically with each other. We briefly review the results and discuss the future prospects.     

Distribution of I, II and III subtypes of voltage-sensitive Na+ channel mRNA in the rat brain

We examined the expression of the I, II and III subtypes of voltage-sensitive Na+ channel mRNA in the rat brain using in situ hybridization histochemistry with oligonucleotide probes. The distribution of cells with strongly positive signals was characteristic for each subtype. Synthesis of each subtype of Na+ channel protein may be regulated by differential mRNA expression.     

The role of valence on the high-affinity binding of Griffonia simplicifolia isolectins to type A human erythrocytes

The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).     

In vivo phosphorylation of the epithelial sodium channel

The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (alpha, beta, and gamma) of ENaC. We found that beta and gamma, but not the alpha subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the beta and gamma subunits in their carboxyl termini, but none of these agents induced de novo phosphorylation of alpha subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the beta and gamma subunits.     

Functional consequences of lidocaine binding to slow-inactivated sodium channels

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use-dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to -20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow-inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow-inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.     

Solution structure of the sodium channel inactivation gate

The sodium channel initiates action potentials by opening in response to membrane depolarization. Fast channel inactivation, which is required for proper physiological function, is mediated by a cytoplasmic loop proposed to occlude the ion pore via a hinged lid mechanism with the triad IFM serving as a hydrophobic "latch". The NMR solution structure of the isolated inactivation gate reveals a stably folded core comprised of an alpha-helix capped by an N-terminal turn, supporting a model in which the tightly folded core containing the latch motif pivots on a more flexible hinge region to occlude the pore during inactivation. The structure, in combination with substituted cysteine mutagenesis experiments, indicates that the IFM triad and adjacent Thr are essential components of the latch and suggests differing roles for the residues of the IFMT motif in fast inactivation.     

Genetic analysis of the epithelial sodium channel in Liddle's syndrome

BACKGROUND: Liddle's syndrome is an autosomal inheritable disorder that causes hypertension due to excess function of sodium channel. OBJECTIVE: To analyze the DNA sequence of the amiloride-sensitive epithelial sodium channel (ENaC) in three patients who had low-renin hypertension with hypokalemia. The patients included a 24-year-old woman and her 20-year-old brother whose mother was hypertensive. The third patient was a 15-year-old girl with no family history of hypertension. METHODS: The DNA sequence of the ENaC was analyzed as follows. Venous blood samples were collected from the patients and total genomic DNA was prepared by standard methods. Specific primers were used for direct polymerase chain reaction; one set of primers for amplifying the C terminus (codon 523-638) of the , subunit of ENaC, and two sets of primers for amplifying the C terminus (codons 525-587 and 568-650) of the y subunit of ENaC. Polymerase chain reaction products were purified and subjected to direct DNA sequence analysis. RESULTS: Direct sequence analysis demonstrated the presence of a single-base substitution in one segment of the 0 subunit of ENaC, a C-T transition that changed the encoded Pro (CCC) at codon 616 to Ser (TCC) in the siblings (cases 1 and 2). In case 3, we found a missense mutation of Pro (CCC) to Leu (CTC) at codon 616. Case 3 is considered to be sporadic, since DNA sequencing of the PY motif of her parents gave normal results. CONCLUSIONS: The DNA sequences of the ENaC in three patients with Liddle's syndrome were analyzed. In one family case, we found a new missense mutation of Pro (CCC) to Ser (TCC) at codon 616 in the 0 subunit of ENaC. A genetic analysis of the amiloride-sensitive epithelial sodium channel is recommended in assessing patients with low-renin, salt-sensitive hypertension whose blood pressure is not responsive to spironolactone treatment.