Coexpression and characterization of the human large-conductance Ca2+-activated K+ channel α + β1 subunits in HEK293 cells

Large-conductance Ca²⁺-activated K⁺ channel is formed by a tetramer of the pore-forming α-subunit and distinct accessory β-subunits (β1–β4) which contribute to BK_Ca channel molecular diversity. Accumulative evidences indicate that not only α-subunit alone but also the α + β subunit complex and/or β-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of α + β1 subunit complex or β1-subunit in BK_Ca channel, we established an expression system that reliably coexpress hSloα + β1 subunit complex in HEK293 cells. The coexpression of hSloα + β1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloα + β1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloα + β1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca²⁺-sensitivity, voltage-dependency, high conductance (151 ± 7 pS), and its pharmacological activation and inhibition.     

Restoration of phosphorylation capacity to the dormant half of the α-subunits of Na, K-ATPase

Purified kidney Na⁺,K⁺-ATPase whose α-subunit is cleaved by chymotrypsin at Leu²⁶⁶-Ala²⁶⁷, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of α-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the α-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of α-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all α-subunits.     

Asymmetric orientation of amino groups in the α-subunit and the β-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

The orientation of amino groups in the membrane in the α- and β-subunits of (Na⁺ + K⁺)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[¹²⁵I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na⁺ + K⁺)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na⁺ + K⁺)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of ¹²⁵I-labeling within the α-subunit. The preservation of (Na⁺ + K⁺)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E₁- and E₂-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.     

Protective effect of Na+ and K+ against inactivation of (Na+ + K+-ATPase by high concentrations of 2-mercaptoethanol at high temperatures

Purified dog kidney (Na⁺ + K⁺)-ATPase (EC 3.6.1.3) was inactivated with high concentrations of 2-mercaptoethanol at 50–55°C. The inactivation was prevented by NaCl or KCl, with KCl being more effective than NaCl (the former ion being about one order more efficient under a typical set of experimental conditions). A disulfide bond in the β-subunit of the enzyme protein was prevented from reductive cleavage by NaCl or KCl in accordance with protection of the enzyme activity. Choline chloride did not exert a significant protective effect over a similar concentration range. (Na⁺ + K⁺)-ATPase was also inactivated with high concentrations of 2-mercaptoethanol in the presence of low concentrations of dodecyl sulfate. This inactivation was also prevented by NaCl or KCl, with the latter being again more efficient than the former. These results indicate that Na⁺ and K⁺ bound to their respective ion-binding sites on the α-subunit exert a protective effect on a disulfide bond on the β-subunit. This suggests some sort of interaction between the α- and the β-subunits.     

Polarized distribution of Na, K-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na⁺, K⁺-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na⁺, K⁺-ATPase activity 2.6-fold more sensitive to ouabain (I₅₀=1.0±0.1 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). As depicted in [³H]ouabain binding experiments, when the [³H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na⁺, K⁺-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na⁺, K⁺-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Covalent Cross-Linking of Radiolabeled Human Chorionic Gonadotropin to Rat Ovarian Luteinizing Hormone Receptor with Glutaraldehyde

Abstract

Crude plasma membranes of pseudopregnant rat ovaries were incubated with ¹²⁵I-labeled human chorionic gonadotropin (¹²⁵I-hCG) and the formed luteinizing hormone (LH)/hCG receptor-¹²⁵I-hCG complex was solubilized, partially purified by Sepharose 6B gel filtration, cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. An apparent molecular weight (mol wt) of 130,000 was obtained for the non-reduced complex. A similar-sized complex was observed, when ³H-hCG (β-subunit labeled) instead of ¹²⁵I-hCG (α-subunit labeled) was used, indicating that the complex contains intact hCG. Upon reduction of the cross-linked receptor-¹²⁵I-hCG complex, a 105,000 mol wt complex in addition to the 130,000 one was observed. No smaller complexes appeared, however, upon reduction of the receptor-³H-hCG complex, suggesting that the α-subunit of hCG predominantly interacts with the receptor. The cross-linking efficiency was dependent on protein concentration, glutaraldehyde concentration, pH, reaction time and temperature. Under optimal conditions (2 mM glutaraldehyde, pH 7.0-8.0, 60 min, 20°C) no nonspecific complexes appeared and the efficiency of cross-linking of the partially purified detergent-solubilized receptor-¹²⁵I-hCG complex was approximately 30%. Glutaraldehyde thus provides a rapid and efficient cross-linking reagent to covalently attach ¹²⁵I-hCG to its receptor and is likely to be highly applicaple to other receptor-ligand systems as well.     

Large conductance Ca2+-activated K+ channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects

Large conductance calcium activated potassium channels (BK_Ca) are fundamental in the control of cellular excitability. Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BK_Ca channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BK_Ca channel α-subunit alone or α + β1-subunit complex.Channel activity was determined using a non-radioactive Rb⁺ efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb⁺ efflux both in cells expressing α-subunit alone or α + β1-subunits. Co-expression of the β1-subunit modified the Rb⁺ efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC₄₀ values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BK_Ca channel α + β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC₄₀ values when tested on the BK_Ca α + β1-subunit expressing cells compared to BK_Ca α-subunit expressing cells. Further, the E_max values for BKPr4, BKVV and BKH1 were lower in the BK_Ca channel α + β1-subunit expressing cells.In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BK_Ca channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.     

Cryo-EM Structures of Azospirillum brasilense Glutamate Synthase in Its Oligomeric Assemblies

Bacterial NADPH-dependent glutamate synthase (GltS) is a complex iron–sulfur flavoprotein that catalyzes the reductive synthesis of two L-Glu molecules from L-Gln and 2-oxo-glutarate. GltS functional unit hosts an α-subunit (αGltS) and a β-subunit (βGltS) that assemble in different αβ oligomers in solution. Here, we present the cryo-electron microscopy structures of Azospirillum brasilense GltS in four different oligomeric states (α₄β₃, α₄β₄, α₆β₄ and α₆β₆, in the 3.5- to 4.1-Å resolution range). Our study provides a comprehensive GltS model that details the inter-protomeric assemblies and allows unequivocal location of the FAD cofactor and of two electron transfer [4Fe–4S]^+1,+2 clusters within βGltS.     

Sensitivity of oocyte-expressed epithelial Na channel to glibenclamide

The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of α-, β- and γ-subunits and the α- and β-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat α- (rα) or guinea-pig α-subunit (gpα) with Xenopus βγ-subunits. The gpαxβγ was significantly stimulated by glibenclamide (100 μM) (184±15%), whereas the rα-combination was slightly down-regulated by the sulfonylurea (79±4%). The stimulating effect did not interfere with Na⁺-self-inhibition resulting from intracellular accumulation of Na⁺-ions. We exchanged cytosolic termini between both orthologs but the gpα-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-β-S but not by ATP-γ-S, when applied intracellularly. Intracellular loading with Na⁺-ions after inhibition of Na⁺/K⁺-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH₃) slightly reduced stimulation of I_ami (118±12%; control: 132±9%) while phosphatidylinositol-4,5-biphosphate (PIP₂) significantly reduced the effect of glibenclamide to 101±3%.     

Na+,K+-ATPase: on the nature of the "cross-linked" subunits obtained in the presence of o-phenanthroline and cupric ion.

In previous studies on the quaternary structure of Na⁺,K⁺-ATPase, cupric-phenanthroline complex (CP) has been used for the cross-linking of the enzyme subunits. Here we show that the same products obtained in the presence of CP (α,α-dimer, α,β-dimer, and products of higher molecular weight) are also obtained when the enzyme is exposed to Cu²⁺ without o-phenanthroline. The α,β-dimer (but not the α,α-dimer) is dissociated in the presence of EDTA; indicating that this product is not the result of the covalent cross-linking of the subunits through a disulfide bond. The nature of the α,α-dimer remains to be determined. The findings suggest that the results of “cross-linking” experiments with CP should be interpreted with caution until the products are more clearly identified.     

ATP4A gene regulatory network for fine-tuning of proton pump and ion channels

The ATP4A encodes α subunit of H⁺, K⁺-ATPase that contains catalytic sites of the enzyme forming pores through cell membrane which allows the ion transport. H⁺, K⁺-ATPase is a membrane bound P-type ATPase enzyme which is found on the surface of parietal cells and uses the energy derived from each cycle of ATP hydrolysis that can help in exchanging ions (H⁺, K⁺ and Cl^?) across the cell membrane secreting acid into the gastric lumen. The 3-D model of α-subunit of H⁺, K⁺-ATPase was generated by homology modeling. It was evaluated and validated on the basis of free energies and amino acid residues. The inhibitor binding amino acid active pockets were identified in the 3-D model by molecular docking. The two drugs Omeprazole and Rabeprazole were found more potent interactions with generated model of α-subunit of H⁺, K⁺-ATPase on the basis of their affinity between drug–protein interactions. We have generated ATP4A gene regulatory networks for interactions with other proteins which involved in regulation that can help in fine-tuning of proton pump and ion channels. These findings provide a new dimension for discovery and development of proton pump inhibitors and gene regulation of the ATPase. It can be helpful in better understanding of human physiology and also using synthetic biology strategy for reprogramming of parietal cells for control of gastric ulcers.     

Species-specific peculiarities of functional reactions of the sodium pump to phosphorylation by protein kinase A

The Na⁺,K⁺-ATPase isolated from shark rectal gland or pig kidney was inserted into liposomes and phosphorylated with cAMP-dependent protein kinase without detergent. Stoichiometry of phosphorylation of α-subunit of the enzyme was 0.9 and 0.2 mol P_i/mol α-subunit of the pig kidney and shark gland, respectively. The phosphorylation of the shark Na⁺,K⁺-ATPase led to an increase in maximum of hydrolytic activity dependent on the cytoplasmic sodium concentration and the extracellular activation with potassium ions. On the contrary, the phosphorylation of the sodium pump of the pig kidney did not produce any significant functional effect.     

The NH2-terminus of K-Cl cotransporter 3a is essential for up-regulation of Na,K-ATPase activity

K⁺-Cl⁻ cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na⁺,K⁺-ATPase α1-subunit (α1NaK), accompanying significant increases of the Na⁺,K⁺-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous α1NaK inducing no change of the Na⁺,K⁺-ATPase activity. A KCC inhibitor attenuated the Na⁺,K⁺-ATPase activity in rat gastric mucosa in which KCC3a is predominantly expressed, while it had no effects on the Na⁺,K⁺-ATPase activity in rat kidney in which KCC3b is predominantly expressed. In these tissue samples, KCC3a co-immunoprecipitated with α1NaK, while KCC3b did not. Our results suggest that the NH₂-terminus of KCC3a is a key region for association with α1NaK, and that KCC3a but not KCC3b can regulate the Na⁺,K⁺-ATPase activity.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na⁺ and K⁺ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic α-subunit (four isoforms) and an ancillary β-subunit (three isoforms). Mutations in the α₂-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase α₂-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [³H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. ⁸⁶Rb⁺-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase α₂-isoform.     

Substitutions in the cardenolide binding site and interaction of subunits affect kinetics besides cardenolide sensitivity of insect Na,K-ATPase

Substitutions within the cardenolide target site of several insects' Na,K-ATPase α-subunits may confer resistance against toxic cardenolides. However, to which extent these substitutions alter the Na,K-ATPase's kinetic properties and how they interact with different β-subunits is not clear. The cardenolide-adapted milkweed bug Oncopeltus fasciatus possesses three paralogs of the α-subunit (A, B, and C) that differ in number and identity of resistance-conferring substitutions. We introduced these substitutions into the α-subunit of Drosophila melanogaster and combined them with the β-subunits Nrv2.2 and Nrv3. The substitutions Q111T-N122H-F786N-T797A (A-copy mimic) and Q111T-N122H-F786N (B-copy mimic) mediated high insensitivity to ouabain, yet they drastically lowered ATPase activity. Remarkably, the identity of the β-subunit was decisive and all α-subunits were less active when combined with Nrv3 than when combined with Nrv2.2. Both the substitutions and the co-expressed β-subunit strongly affected the enyzme's affinity for Na⁺ and K⁺. Na⁺ affinity was considerably higher for all enzymes expressed with nrv3 while expression with nrv2.2 mostly increased K⁺ affinity. Our results provide the first evidence that resistance against cardenolides comes at the cost of significantly altered kinetic properties of the Na,K-ATPase. The β-subunit can strongly modulate these properties but cannot fully compensate for the effect of the substitutions.     

Identification,purification and partial characterization of low molecular weight protein inhibitor of Na+/K+-ATPase from pulmonary artery smooth muscle cells

We have identified a novel endogenous low mol wt. (15.6 kDa) protein inhibitor of Na⁺/K⁺-ATPase in cytosolic fraction of bovine pulmonary artery smooth muscle cells. The inhibitor showed different affinities toward the α₂β₁ and α₁β₁ isozymes of Na⁺/K⁺-ATPase, where α₂ is more sensitive than α₁. The inhibitor interacted reversibly to the E₁ site of the enzyme and blocked the phosphorylated intermediate formation. Circular dichroism study suggests that the inhibitor causes an alteration in the confirmation of the enzyme.     

Facile “stop codon” method reveals elevated neuronal toxicity by discrete S87p-α-synuclein oligomers

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to α-synuclein (α-Syn). Three different α-Syn species phosphorylated at Serine 87 (S87p-α-Syn), Serine 129 (S129p-α-Syn) and Serine 87/129 (S87p,129p-α-Syn) were prepared through the ‘stop codon’ method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated α-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu²⁺ ions and pyridoxal 5′-phosphate. Aggregation trends induced by DA or DA/Cu²⁺ were similar between phosphorylated and non-phosphorylated α-Syn in SDS–PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated α-Syn (Np-α-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-α-Syn and S87p,129p-α-Syn oligomers induced by DA/Cu²⁺, showed higher toxicity than did S129p-α-Syn. When α-Syn was treated with pyridoxal 5′-phosphate in the presence of DA or Cu²⁺ to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. α-Syn co-incubated with DA or DA/Cu²⁺ showed less cellular toxicity upon pyridoxal 5′-phosphate treatment, especially in the case of DA-induced Np-α-syn. This study supports that phosphorylated oligomers of α-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5′-phosphate can be used as an inhibitor for α-Syn aggregation.     

Heterooligomers of the muscarinic receptor and G proteins purified from porcine atria

Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Gα_o, Gα_i1-3, and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, α-subunits, and caveolins in various combinations. Homooligomers of α_i2 were detected on Western blots, and heterooligomers of α_i2 and α_o were identified by coimmunoprecipitation; thus, a complex may contain at least two α-subunits. Other combinations of α-subunit were not detected. The ratio of total α-subunit to receptor was near 1, as measured by [³⁵S]GTPγS and the antagonist [³H]quinuclidinylbenzilate, and the binding of [³⁵S]GTPγS was manifestly biphasic. The ratio of α_o to α_i1,2 also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of α-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of α-subunit and the receptor.