Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Diversity and Developmental Expression of L-type Calcium Channel ��2 Proteins and Their Influence on Calcium Current in Murine Heart

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVβ2 proteins are the major CaVβ components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVβ3 proteins are also detectable. At least two CaVβ2 variants of ∼68 and ∼72 kDa are expressed. To identify the underlying CaVβ2 variants, cDNA libraries were constructed from poly(A)⁺ RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVβ2 cDNA clones coding for four types of CaVβ2 proteins only differing in their 5′ sequences. CaVβ2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVβ2 protein pattern in vitro. Significant L-type Ca²⁺ currents (I_Ca) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVβ2. Current kinetics were determined by the type of CaVβ2 protein, with the ∼72-kDa CaVβ2a-N1 shifting the activation of I_Ca significantly to depolarizing potentials compared with the other CaVβ2 variants. Inactivation of I_Ca was accelerated by CaVβ2a-N1 and -N4, which also lead to slower activation compared with CaVβ2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVβ2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVβ2a-N1-induced changes of I_Ca kinetics might be essential in embryonic heart.Cardiac contractions require Ca²⁺ influx in cardiomyocytes from the extracellular fluid, which leads to Ca²⁺ release from the sarcoplasmic reticulum via ryanodine receptors (1).This Ca²⁺-induced Ca²⁺ release (CICR)⁴ causes a marked increase in intracellular Ca²⁺ concentration for short periods of time and underlies cardiac contraction (2, 3). The Ca²⁺ influx into cardiac myocytes is mediated by high voltage-activated L-type Ca²⁺ channels (LTCCs), which are heteromultimeric complexes comprised predominantly of the pore-forming CaVα₁ subunit and the auxiliary CaVβ subunit (4). In heart, the principal CaVα₁ subunit, CaVα₁c (CaV1.2), is encoded by the Cacna1C gene (5). Four genes (Cacnb1-4) encoding CaVβ subunits have been identified that are expressed in the heart of different species including human, rabbit, and rat (6, 7, 8).CaVβ proteins are ∼500 amino acid cytoplasmic proteins that bind to the CaVα₁ I-II intracellular loop (9) and affect channel gating properties (4), trafficking (10, 11), regulation by neurotransmitter receptors through G-protein βγ subunit activation (12), and sensitivity to drugs (13). The CaVβ primary sequence encodes five domains, arranged V1-C1-V2-C2-V3. V1, V2, and V3 are variable domains, whereas C1 and C2 are conserved (14). Structural studies reveal that C1 and C2 form a SH3 domain (Src homology 3 domain) and a NK domain (nucleotide kinase domain), respectively (15). Although C1-V2-C2 makes the CaVβ core, in heart the V1 region appears critical for the kinetics of I_Ca and heart function. Accordingly a mutation in the V1 region of the Cacnb2 gene was recently identified as an underlying cause of Brugada syndrome (16).In mice-targeted deletion of the Cacnb2 gene (17) but not of Cacnb1 (18), Cacnb3 (19, 20), or Cacnb4 (21) leads to a morphologically and functionally compromised heart, which causes severe defective remodeling of intra- and extra-embryonic blood vessels and death at early embryonic stages both when the Cacnb2 gene was targeted globally or in a cardiac myocyte-specific way (17). Although these results point to an essential role of CaVβ2 for I_Ca and cardiac function, the existence of various CaVβ2 splice variants and heterogeneity of the expressed CaVβ2 proteins require further studies on the subunit composition of LTCCs in the mouse heart. In addition and in view of the growing number of preclinical studies using mouse models carrying definite Ca²⁺ channel subunits as transgenes in heart tissue, the identification of the relevant gene products underlying the endogenous mouse cardiac L-type channel is essential. Recent mouse models (e.g. 22, 23, 24) carrying a rat CaVβ2 splice variant (“rat CaVβ2a”) (25) expressed in rat and rabbit brain (26), but not in rabbit heart (26), have only escalated this requirement, because it has never been shown that the mouse orthologue of this variant is endogenously expressed in the mouse heart.So far, five CaVβ2 variants varying only in the V1 domain have been identified from different species (25, 27, 28) and in human heart these variants have been obtained mainly by RT-PCR approaches (29, 30). In contrast, there is little information on the CaVβ proteins present in mouse heart, their respective splice variants, and expression ratios. We therefore started to study CaVβ expression in the murine heart using Western blots and cDNA cloning and to reveal their functional impact on LTCCs formed by the murine CaV1.2 protein.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.     

Transient Oxidative Stress Damages Mitochondrial Machinery Inducing Persistent ��-Cell Dysfunction

Transient exposure of β-cells to oxidative stress interrupts the transduction of signals normally coupling glucose metabolism to insulin secretion. We investigated putative persistence of effects induced by one transient oxidative stress (200 μm H₂O₂, 10 min) on insulin secreting cells following recovery periods of days and weeks. Three days after oxidative stress INS-1E cells and rat islets exhibited persistent dysfunction. In particular, the secretory response to 15 mm glucose was reduced by 40% in INS-1E cells stressed 3 days before compared with naïve cells. Compared with non-stressed INS-1E cells, we observed reduced oxygen consumption (−43%) and impaired glucose-induced ATP generation (−46%). These parameters correlated with increased mitochondrial reactive oxygen species formation (+60%) accompanied with down-regulation of subunits of the respiratory chain and decreased expression of genes responsible for mitochondrial biogenesis (TFAM, −24%; PGC-1α, −67%). Three weeks after single oxidative stress, both mitochondrial respiration and secretory responses were recovered. Moreover, such recovered INS-1E cells exhibited partial resistance to a second transient oxidative stress and up-regulation of UCP2 (+78%) compared with naïve cells. In conclusion, one acute oxidative stress induces β-cell dysfunction lasting over days, explained by persistent damages in mitochondrial components.Pancreatic β-cells are poised to sense blood glucose to regulate insulin exocytosis and thereby glucose homeostasis. The conversion from metabolic signals to secretory responses is mediated through mitochondrial metabolism (1). Failure of the insulin secreting β-cells, a common characteristic of both type 1 and type 2 diabetes, derives from various origins, among them mitochondrial impairment secondary to oxidative stress is a proposed mechanism (2).Oxidative stress is characterized by a persistent imbalance between excessive production of reactive oxygen species (ROS)³ and limited antioxidant defenses. Examples of ROS include superoxide (O₂^−̇), hydroxyl radical (OH^⋅), and hydrogen peroxide (H₂O₂). Superoxide can be converted to less reactive H₂O₂ by superoxide dismutase (SOD) and then to oxygen and water by catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin, which constitute antioxidant defenses. Increased oxidative stress and free radical damages have been proposed to participate in the diabetic state (3). In type 1 diabetes, ROS are implicated in β-cell dysfunction caused by autoimmune reactions and inflammatory cytokines (4). In the context of type 2 diabetes, excessive ROS could promote deficient insulin synthesis (5, 6) and apoptotic pathways in β-cells (5, 7). Of note, ROS fluctuations may also contribute to physiological control of cell functions (8), including the control of insulin secretion (9). It should also be stressed that metabolism of physiological nutrient increases ROS without causing deleterious effects on cell function. However, uncontrolled increase of oxidants, or reduction of their detoxification, leads to free radical-mediated chain reactions ultimately triggering pathogenic events. Pancreatic β-cells are relatively weak in expressing free radical-quenching enzymes SOD, CAT, and GPx (10, 11), rendering those cells particularly susceptible to oxidative attacks (12). Mitochondria are not only the main source of cellular oxidants, they are also the primary target of ROS (13, 14).Mitochondria are essential for pancreatic β-cell function, and damages to these organelles are well known to markedly alter glucose-stimulated insulin secretion (15). The mitochondrial genome constitutes one of the targets, encoding for 13 polypeptides essential for the integrity of electron transport chain (16). Damages to mitochondrial DNA (mtDNA) induce mutations that in turn may favor ROS generation, although the contribution of mtDNA mutations to ROS generation remains unclear. We previously reported that patient-derived mitochondrial A3243G mutation, causing mitochondrial inherited diabetes, is responsible for defective mitochondrial metabolism associated with elevated ROS levels and reduced antioxidant enzyme expression (17). On the other hand, mtDNA mutator mice exhibit accelerated aging without changes in superoxide levels in embryonic fibroblasts (18), showing that ROS generation can be dissociated from mtDNA mutations.In humans, mitochondrial defects typically appear with aging (19), accompanied by sustained ROS generation and progressive oxidant-induced damages (20). In support of this “mitochondrial theory of aging” (21), accumulating evidence shows that in older individuals mitochondria are altered, both morphologically and functionally (22). These age-related mitochondrial changes are foreseen to play a role in the late onset diabetes. In a rat model of intrauterine growth retardation, a vicious cycle between accumulation of mtDNA mutations and elevation of ROS production has been associated to β-cell abnormalities and the onset of type 2 diabetes in adulthood (23). Similarly, mitochondrion-derived ROS impair β-cell function in the Zucker diabetic fatty rat (24). Altogether, these observations point to ROS action as a triggering event inducing mitochondrial dysfunction and ultimately resulting in the loss of the secretory response in β-cells (14).In vitro, oxidative stress applied to β-cells rapidly interrupts the transduction of signals normally coupling glucose metabolism to insulin secretion (12, 25). Specifically, we reported that INS-1E β-cells and rat islets subjected to a 10-min H₂O₂ exposure exhibit impaired secretory response associated with mitochondrial dysfunction appearing already during the first minutes of oxidative stress (12). In the context of the mitochondrial theory of aging (21, 26), it is important to know whether transient exposure to H₂O₂ could possibly induce persistent modifications of mitochondrial function. Cells surviving an oxidative stress might carry defects leading to progressive loss of β-cell function. In the present study, we asked the simple but unanswered question if a short transient oxidative stress could induce durable alterations of the mitochondria and thereby chronically impair β-cell function. INS-1E β-cells and rat islets were transiently exposed to H₂O₂ for 10 min and analyzed after days and weeks of standard tissue culture.     

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion ��-Toxins

The scorpion α-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na_vs) and is inactive at insect Na_vs. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa_v1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved “core domain” (residues of the loops connecting the secondary structure elements of the molecule core), and a variable “NC domain” (five-residue turn and the C-tail) as was reported for other scorpion α-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhαIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe¹⁵ of the core domain in toxin potency at rNa_v1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion α-toxins we were able to eliminate the activity of Lqh2 at rNa_v1.4 (skeletal muscle), hNa_v1.5 (cardiac), and rNa_v1.6 channels, with no hindrance of its activity at Na_v1.1–1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.The pivotal role of voltage-gated sodium channels (Na_vs)⁴ in excitability mark them as major targets for a large variety of toxins that bind at distinct receptor sites and modify their gating (1). These channels are large membrane proteins made of a pore-forming α-subunit of ∼260 kDa and auxiliary β-subunits of ∼30 kDa. The α-subunit is composed of four homologous domains (D1–D4), each consisting of six α-helical transmembrane segments (S1–S6) connected by intracellular and extracellular loops. A key feature in Na_vs function is their ability to rapidly activate and inactivate, leading to transient increase in Na⁺ conductance through the cell membrane. This mechanism is attributed to the ability of the positively charged S4 voltage sensors to move across the membrane in response to changes in membrane potential (1, 2).In mammals, at least nine genes encode a variety of Na_v subtypes (1, 3), whose expression varies greatly in different tissues (Na_v1.1–1.3 mainly in the central nervous system; Na_v1.6 in both central and peripheral neurons; Na_v1.7 in the peripheral nervous system; Na_v1.8 and Na_v1.9 in sensory neurons; Na_v1.4 and Na_v1.5 in skeletal and cardiac muscles, respectively). Na_v subtypes are distributed heterogeneously in the human brain and their expression is regulated under developmental and pathological conditions (1, 3–5). In addition, many disorders in humans result from abnormal function due to mutations in various Na_v genes (6–8). Thus, ligands that show specificity for Na_v subtypes may be used for their identification at various tissues and as leads for design of specific drugs. This requires that the bioactive surfaces of these ligands be resolved along with molecular details that determine their specificity.Among the wide range of Na_v modifiers, those derived from scorpion venoms play an important role in studying channel activation (β-toxins) and inactivation (α-toxins) (9–11). The channel site of interaction with scorpion α-toxins, named neurotoxin receptor site-3 (12), is shared also by structurally unrelated toxins from sea anemone and spider venoms (13, 14), which raises questions as to its architecture and boundaries. Based on the findings that site-3 toxins eliminate a gating charge component associated with the movement of D4/S4 (15, 16), and that this segment plays a critical role in coupling channel inactivation to activation (17), scorpion α-toxins were postulated to inhibit channel inactivation by hindering the outward movement of this segment during depolarization (9).Scorpion α-toxins constitute a class of structurally and functionally related 61–67-residue long polypeptides reticulated by four conserved disulfide bridges. Despite a common βαββ core (10, 18, 19) these toxins are highly diverse in sequence and preference for insect and mammalian Na_vs. Indeed, the α-toxin class is divided to pharmacological groups according to their toxicity in insects and mice brain and ability to compete on binding at insect and mammalian Na_vs (10) (supplemental Fig. S1): (i) classical anti-mammalian toxins, such as Aah2 (from Androctonus australis hector) and Lqh2 (from Leiurus quinquestriatus hebraeus), which bind with high affinity to Na_vs at rat brain synaptosomes and are practically non-toxic to insects; (ii) α-toxins, such as LqhαIT, which strongly affect insect Na_vs and are weak in mammalian brain; and (iii) α-like toxins, such as Lqh3 and BmKM1 (from Buthus martensii Karsch), which are active in both mammalian brain and insects.Efforts to identify α-toxin residues involved in the interaction with the Na_v receptor site-3 revealed a generally common bioactive surface divided to two topologically distinct domains: a conserved “core domain” formed by residues of the loops connecting the secondary structure elements of the molecule core, and a variable “NC domain” formed by the five-residue turn (residues 8–12) and the C-tail (20–23). These analyses raised the hypothesis that a protruding conformation of the NC domain correlates with high activity at insect Na_vs, whereas a flat conformation of this domain appears in α-toxins active at the brain channel rNa_v1.2a (21). The correlation of this structural difference with toxin preference for Na_v subtypes was corroborated by constructing the bioactive surface of LqhαIT on the scaffold of the anti-mammalian α-toxin Aah2 ending up with a chimera (Aah2^{LqhαIT(face)}) active on insects, whose NC domain is in the protruding conformation (21). Despite this result, the molecular requirements that enable high affinity binding of classical α-toxins to mammalian Na_vs have not been clarified, and only initial data about the channel region that constitutes receptor site-3 is available (Refs. 24–26; also see Ref. 10 for review).Lqh2 is a 64-residue long toxin from L. quinquestriatus hebraeus (Israeli yellow scorpion) (27) that is almost identical in sequence (96% identity) to the most active anti-mammalian toxin, Aah2, whose structure and action are documented (18, 28, 29). By functional expression and mutagenesis we uncovered residues on the Lqh2 exterior that are putatively involved in bioactivity. By construction of these residues on the scaffold of the anti-insect toxin LqhαIT we confirmed their bioactive role and differentiated those that determine toxin potency from those contributing to toxin efficacy. Comparison to other α-toxins was then instrumental for the design of an Lqh2 mutant that exhibits high specificity for the neuronal channels hNa_v1.1, rNa_v1.2a, and rNa_v1.3.     

Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-α1 or CFP-NKA-α2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 ± 3.4% for NKA-α1 and 27.5 ± 2.5% for NKA-α2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-α2 versus PLM-NKA-α1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving ≥3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-α1 and NKA-α2 function. Isoproterenol enhanced internal Na⁺ affinity of both isoforms (K_½ decreased from 18.1 ± 2.0 to 11.5 ± 1.9 mm for NKA-α1 and from 16.4 ± 2.5 to 10.4 ± 1.5 mm for NKA-α2) without altering maximum transport rate (V_max). Protein kinase C activation also decreased K_½ for both NKA-α1 and NKA-α2 (to 9.4 ± 1.0 and 9.1 ± 1.1 mm, respectively) but increased V_max only for NKA-α2 (1.9 ± 0.4 versus 1.2 ± 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-α1 and NKA-α2 in a comparable but not identical manner.Cardiac Na/K-ATPase (NKA)³ regulates intracellular Na⁺, which in turn affects intracellular Ca²⁺ and contractility via Na⁺/Ca²⁺ exchange. Members of the FXYD family of small, single membrane-spanning proteins, including phospholemman (PLM) and the NKA γ-subunit (1), have emerged recently as tissue-specific regulators of NKA. PLM is the only FXYD protein known to be highly expressed in cardiac myocytes and is also unique within the family in that it is phosphorylated at two or more sites by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (2, 3). In the heart, PLM is a major phosphorylation target for both PKA and PKC.Co-immunoprecipitation experiments have demonstrated that PLM is physically associated with NKA (4–8), and this is not affected by PLM phosphorylation (6, 7). We have shown recently (9) that PLM and NKA are in very close proximity, such that fluorescence resonance energy transfer (FRET) occurs. PLM phosphorylation by either PKA or PKC reduces the FRET significantly, suggesting that although PLM and NKA are not physically dissociated upon phosphorylation, their interaction is altered. PLM inhibits NKA (4, 8, 10, 11), mostly by reducing the affinity of the pump for internal Na⁺. PLM phosphorylation relieves this inhibition and thus mediates the enhancement of NKA function by α- and β-adrenergic stimulation in mouse ventricular myocytes (10, 11).There are multiple NKA isoforms in cardiac myocytes. NKA-α1 is the dominant, ubiquitous isoform, whereas NKA-α2 and NKA-α3 are present in relatively small amounts and in a species-dependent manner (12). For instance, the adult rodent heart expresses NKA-α1 and NKA-α2, although dogs and monkeys do not have the NKA-α2 subunit (13). In humans all three NKA-α isoforms can be detected (14). It has been suggested that NKA-α2 and NKA-α3 are located mainly in the T-tubules, at the junctions with the sarcoplasmic reticulum, where they could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. There is rather convincing evidence supporting such a model in the smooth muscle (15). However, things are less clear in the heart. The functional density of NKA-α2 is significantly higher in the T-tubules (versus external sarcolemma) in cardiac myocytes from both rats (16, 17) and mice (18), but their precise localization with respect to the junctions with the sarcoplasmic reticulum is not known. Based on Ca²⁺ transients from heterozygous NKA-α1^+/− and NKA-α2^+/− mice, James et al. (19) concluded that NKA-α2 is involved in cardiac myocyte Ca²⁺ regulation, whereas NKA-α1 is not. Further support for this idea came from the observation that replacing mouse NKA-α2 with a low affinity mutant leads to a loss of glycoside inotropy (20), and increased expression of NKA-α2 decreased the Na⁺/Ca²⁺ exchange current and Ca²⁺ transients (21). However, other findings challenge the preferential role of NKA-α2 in regulating intracellular Ca²⁺ and contractility. Moseley et al. (22) showed that NKA-α1^+/− mice were severely compromised, and Dostanic et al. (23) showed that NKA-α1 is also physically and functionally associated with the Na⁺/Ca²⁺ exchanger.In this context, it is important to determine whether NKA-α1 and NKA-α2 interact differently with PLM. The data available so far on this are contradictory. We have found (7) that NKA-α1, NKA-α2, and NKA-α3 isoforms co-immunoprecipitate PLM, both unphosphorylated and phosphorylated, in rabbit heart. In contrast, Silverman et al. (8) reported that NKA-α1 but not NKA-α2 co-immunoprecipitate with PLM in ventricular myocytes from guinea pig. The functional data are also contradictory. PLM was found to reduce the affinity for Na⁺ of both NKA-α1 and NKA-α2 isoforms in a heterologous expression system (4), whereas Silverman et al. (8) reported that forskolin-induced PLM phosphorylation results in a higher NKA-α1-mediated current and no change in the current generated by NKA-α2.Here we used two methods to investigate whether the interaction and functional effects of PLM on NKA are NKA-α isoform-specific. First, we used FRET to assess the interaction between PLM-YFP and CFP-NKA-α1/CFP-NKA-α2 transfected in HEK-293 cells and how PLM phosphorylation by PKA and PKC affects this interaction. Second, we measured NKA function in myocytes isolated from wild-type (WT) mice and mice where NKA isoforms have swapped ouabain affinities (SWAP; NKA-α1 is ouabain-sensitive, whereas NKA-α2 is ouabain-resistant) (23). In this way we could test the effect of β-adrenergic stimulation separately on NKA-α1 and NKA-α2 isoforms in the native myocyte environment, as an indicator of the functional interaction with PLM. Our results indicate that NKA-α1 and NKA-α2 interact similarly with PLM, and this interaction is equally affected by PLM phosphorylation.