Synthesis and initial evaluation of novel,non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS? compound libraries led to the identification of compound 1 as a dual inhibitor of the α_v-integrins α_vβ₃ and α_vβ₅. Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the α_vβ₃ and α_vβ₅ integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.     

The TF1-ATPase and ATPase activities of assembled α3β3γ, α3β3γδ, and α3β3γε complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the α3β3, and α3β3δ complexes

The ATPase activity of the F₁-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 µM where 70% stimulation is observed at 36°C. Half maximal stimulation is observed at about 3 µM dye. At rhodamine 6G concentrations greater than 10 µM, ATPase activity declines with 50% inhibition observed at about 75 µM dye. The ATPase activities of the ₃₃ and ₃₃ complexes assembled from isolated subunits of TF₁ expressed inE. coli deleted of theunc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF₁. In contrast, the ATPase activities of the ₃₃ and ₃₃ complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 µM dye at 36°C. The ATPase activity of TF₁ is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF₁ is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 µM dye at 30°C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF₁ stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.     

Physiological and Pathological Roles of α3β1 Integrin

α3β1 integrin has been considered to be a mysterious adhesion molecule due to the pleiotropy in its ligand-binding specificity. However, recent studies have identified laminin isoforms as high-affinity ligands for this integrin, and demonstrated that α3β1 integrin plays a number of essential roles in development and differentiation, mainly by mediating the establishment and maintenance of epithelial tissues. Furthermore, α3β1 integrin is also implicated in many other biological phenomena, including cell growth and apoptosis, angiogenesis and neural functions. This integrin receptor forms complexes with various other membrane proteins, such as the transmembrane-4 superfamily proteins (tetraspanins), cytoskeletal proteins and signaling molecules. Recently, lines of evidence have been reported showing that complex formation regulates integrin functions in cell adhesion and migration, signal transduction across cell membranes, and cytoskeletal organization. In addition to these roles in physiological processes, α3β1 integrin performs crucial functions in various pathological processes, especially in wound healing, tumor invasion and metastasis, and infection by pathogenic microorganisms.This revised version was published online in June 2005 with a corrected cover date.     

Synthesis of 4-hydroxy-β3-homoprolines and their insertion in α/β/α-tripeptides

The stereoselective syntheses of 2-cyclopropyl- and (2S)-2-hydroxymethyl-(3R,4S)-4-hydroxy-β³-homoproline are described. The reported amino acids were constructed through 1,3-dipolar cycloaddition of strained alkylidenecyclopropanes with enantiopure pyrroline N-oxides derived from malic acid followed by thermal rearrangement of the adducts in the presence of trifluoroacetic acid. The two-step sequence afforded the homoprolines suitably protected to be directly used as building blocks in peptidomimetic synthesis as proved by the synthesis of the two model mixed α/β/α tripeptides Phe-β³-HPro-Val.     

Interaction of αVβ3 and αVβ6 Integrins with Human Parechovirus 1

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to α_V integrins. The interaction of HPEV1 with α_V integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins α_Vβ₃ and α_Vβ₆ to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, α_Vβ₆ bound more efficiently than α_Vβ₃ to immobilized HPEV1. Moreover, soluble α_Vβ₆, but not α_Vβ₃, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.Picornaviruses consist of a positive-sense, single-stranded infectious RNA genome of approximately 7.3 kb enclosed in a capsid composed of 60 copies of each of the three or four capsid proteins (VP1 to VP4). Human parechovirus 1 (HPEV1) is a member of the Parechovirus genus of the Picornaviridae family (38, 70). There are currently eight completely sequenced human parechovirus types and 14 described types (4, 19, 24, 30, 38, 39, 51, 58, 78). In addition, the Parechovirus genus currently has four Ljungan virus members that infect rodents. HPEV1 exhibits several distinct molecular characteristics compared to other picornaviruses (38, 71). These include the lack of the maturation cleavage of the capsid proteins VP0 to VP4 (N-terminal) and VP2 (C-terminal), existence of an approximately 30-amino-acid-long extension to the N terminus of VP3, a unique nonstructural protein 2A, and a 5′ untranslated region that is more closely related to picornaviruses infecting animals than those infecting humans.HPEV infections are common during the first years of life and are often mild or asymptomatic (20, 28, 42, 73, 80). Recently, a number of new types have been identified, and their prevalence in stool samples, for example, highlights their clinical importance. Normally, they cause gastroenteritis and respiratory infections, but severe illnesses, such as infections of the central nervous system, generalized infections of neonates, and myocarditis, have also been associated with HPEV infections (1, 8, 10, 28, 80). Currently, the role of the unique molecular, structural, and antigenic characteristics of HPEVs in the pathogenesis of infection is unknown.HPEV types 1, 2, 4, 5, and 6 are known to possess an RGD motif near the C terminus of VP1 that is known to facilitate binding of cellular ligands (e.g., fibronectin) to α_v integrins. The motif is in an analogous position to motifs in coxsackievirus A9 (CAV9) and echovirus 9 (EV9; Barty strain) (Fig. ​(Fig.1).1). The role of the RGD sequence in cellular entry and subsequent replication of HPEV1 has been shown through blocking assays with RGD-containing peptides, mutation of the sequence, and function-blocking antibodies to α_v integrins (11, 43, 62, 71). These results strongly suggested that α_v integrins play a central role in the initiation of HPEV1 infection. Direct involvement of α_v integrins in the infectious entry of HPEV1 was further confirmed by overexpression of human α_vβ₁ and α_vβ₃ integrins in Chinese hamster ovary (CHO) cells, allowing successful virus infection (74). There are no reports yet on the identification of receptors for the HPEV types lacking the RGD motif (HPEV3, HPEV7, and HPEV8) (19, 39, 51).Open in a separate windowFIG. 1.Sequence alignments. Amino acid sequence alignment of the viral coat protein VP1 from different picornaviruses with the CAV9 secondary structure derived from the atomic model displayed above the alignment (34). The columns boxed in blue with red letters signify similarity, and the red column signifies identity. There is limited similarity between HPEV and other picornaviruses. C-terminal RGD motifs are boxed in red.Although the crystal structures of several picornaviruses have been determined (3, 26, 34, 35, 44, 57, 59, 65, 68, 72) and the receptor interactions have been studied in detail by X-ray crystallography, electron cryo-microscopy (cryo-EM), and three-dimensional (3D) image reconstruction (6, 9, 23, 31, 32, 47, 83), there is no structural information available for the parechoviruses or parechovirus-receptor complexes. Here, we compare the binding of α_Vβ₃ and α_Vβ₆ to HPEV1 in vitro by biochemical assays and determine the structures of HPEV1 and the corresponding HPEV1-integrin complexes.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.     

c-Abl Is an Upstream Regulator of Acid Sphingomyelinase in Apoptosis Induced by Inhibition of Integrins αvβ3 and αvβ5

Inhibition of integrins αvβ3/αvβ5 by the cyclic function-blocking peptide, RGDfV (Arg-Gly-Asp-Phe-Val) can induce apoptosis in both normal cells and tumor cells. We show that RGDfV induced apoptosis in ECV-304 carcinoma cells, increased activity and mRNA expression of acid sphingomyelinase (ASM), and increased ceramides C(16), C(18∶0), C(24∶0) and C(24∶1) while decreasing the corresponding sphingomyelins. siRNA to ASM decreased RGDfV-induced apoptosis as measured by TUNEL, PARP cleavage, mitochondrial depolarization, and caspase-3 and caspase-8 activities, as well as by annexinV in a 3D collagen model. These findings indicate a causal role for ASM in RGDfV-induced apoptosis in ECV-304. We have shown that c-Abl, a non-receptor tyrosine kinase, also mediates RGDfV-induced apoptosis. However, c-Abl, has not been previously linked to ASM in any system. Here we show that STI-571 (imatinib, inhibitor of c-Abl) inhibited RGDfV-induced ASM activity. Furthermore, STI-571 and c-Abl-siRNA both inhibited RGDfV-induced increase in ASM mRNA, but ASM-siRNA did not affect c-Abl phosphorylation or expression, supporting that c-Abl regulates the RGDfV-induced increase in ASM expression. These studies implicate ASM as a mediator of apoptosis induced by inhibition of integrins αvβ3/αvβ5, and for the first time place c-Abl as an upstream regulator of ASM expression and activity.     

Diversity of Structure and Function of α1α6β3δ GABAA Receptors: COMPARISON WITH α1β3δ AND α6β3δ RECEPTORS*

δ subunit-containing γ-aminobutyric acid, type A (GABA_A)receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with α₁ and/or α₆ subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA_A receptor pentamers by concatenation. These receptors composed of α₁, α₆, β₃, and δ subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3α,21-dihydroxy-5α-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that δ can assume multiple positions in a receptor pentamer made up of α₁, α₆, β₃, and δ subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of δ subunits between two α subunits in α₁α₆β₃δ receptors. This property is shared by α₁β₃δ and α₆β₃δ receptors, but there are differences in the additionally expressed isoforms.     

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1

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Interaction of α-N-acetyl-β-endorphin and calmodulin

Acetylation at the α-amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide β-endorphin, α-N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for α-N-acetyl-β-endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like β-endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to β-endorphin, suggest that residues 14–24 exhibit α-helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the α-amino terminal of β-endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, β-endorphin and the α-N-acetylated peptide behave very similarly with respect to calmodulin association.     

Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.     

The α/β Interfaces of α1β1, α3β3, and F1: Domain Motions and Elastic Energy Stored during γ Rotation

ATP synthase (F_oF₁) consists of F₁ (ATP-driven motor) and F_o (H⁺-driven motor). F₁ is a complex of ₃₃ subunits, and is the rotating cam in ₃₃. Thermophilic F₁ (TF₁) is exceptional in that it can be crystallized as a monomer and an ₃₃ oligomer, and it is sufficiently stable to allow refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified or . The nucleotide-dependent open–close conversion of conformation is an inherent property of an isolated and energy and signals are transferred through / interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F₁ (MF₁) and TF₁ were analyzed by an atom search within the limits of 0.40 nm across the interfaces. Seven (plus thermophilic loop in TF₁) contact areas are located at both the catalytic and noncatalytic interfaces on the open form. The number of contact areas on closed increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in . The notion of elastic energy in F_oF₁ has been revised. X-ray crystallography of F₁ is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of F_oF₁. The domain motion and elastic energy in F_oF₁ will be elucidated by time-resolved crystallography.     

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.     

Positional Scanning Mutagenesis of α-Conotoxin PeIA Identifies Critical Residues That Confer Potency and Selectivity for α6/α3β2β3 and α3β2 Nicotinic Acetylcholine Receptors

The nicotinic acetylcholine receptor (nAChR) subtype α6β2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6β2* and α3β2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6β2* and α3β2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3β2β3 and α3β2 receptors. Mutagenesis of PeIA revealed that Asn¹¹ was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn¹¹ with a positively charged amino acid essentially abolished the activity of PeIA for α3β2 but not for α6/α3β2β3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3β2β3 than α3β2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu¹⁵² of the α6 subunit and Lys¹⁵² of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6β2* nAChRs.     

Synthesis and evaluation of bivalent, peptidomimetic antagonists of the αvβ3 integrins

Targeting the integrin α(v)β(3) by directly interfering with its function is considered to be an effective and non-cytotoxic strategy for the treatment of tumor. In this study, a series of bivalent analogs of peptidomimetic integrin antagonists IA 1 and IAC 2 were designed, synthesized, and evaluated for their ability to inhibit the integrin α(v)β(3). All the bivalent ligands exhibited increased potency compared to that of their monomeric counterparts for the integrin α(v)β(3) with low nanomolar range binding affinity. The best bivalent ligand 6 tested in the series has an IC(50)=0.09 nM evaluated by ELISA assay. We conclude that multivalency is providing a useful template for the development novel integrin α(v)β(3) antagonists as potential therapeutics.     

Role of negatively charged amino acids in β4 F-loop in activation and desensitization of α3β4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the β4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the α3β4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at − 40 mV produced inward currents which displayed biphasic desensitization. The EC₅₀ of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC₅₀ increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.     

A Specific and Essential Role for Na,K-ATPase α3 in Neurons Co-expressing α1 and α3

Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na⁺ imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na⁺ concentration ([Na⁺]_i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na⁺]_i, similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na⁺]_i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na⁺]_i is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na⁺. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na⁺ binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic [Na⁺]_i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function.