Lysine-Rich Extracellular Rings Formed by hβ2 Subunits Confer the Outward Rectification of BK Channels

The auxiliary β subunits of large-conductance Ca²⁺-activated K⁺ (BK) channels greatly contribute to the diversity of BK (mSlo1 α) channels, which is fundamental to the adequate function in many tissues. Here we describe a functional element of the extracellular segment of hβ2 auxiliary subunits that acts as the positively charged rings to modify the BK channel conductance. Four consecutive lysines of the hβ2 extracellular loop, which reside sufficiently close to the extracellular entryway of the pore, constitute three positively charged rings. These rings can decrease the extracellular K⁺ concentration and prevent the Charybdotoxin (ChTX) from approaching the extracellular entrance of channels through electrostatic mechanism, leading to the reduction of K⁺ inflow or the outward rectification of BK channels. Our results demonstrate that the lysine rings formed by the hβ2 auxiliary subunits influences the inward current of BK channels, providing a mechanism by which current can be rapidly diminished during cellular repolarization. Furthermore, this study will be helpful to understand the functional diversity of BK channels contributed by different auxiliary β subunits.     

Co-expression of γ2 Subunits Hinders Processing of N-Linked Glycans Attached to the N104 Glycosylation Sites of GABAA Receptor β2 Subunits

GABA_A receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and β subunits with or without a γ2 subunit. The α1β2γ2 receptor is the most abundant GABA_A receptor in brain. Co-expression of γ2 with α1 and β2 subunits causes conformational changes, increases GABA_A receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three β2 subunit glycosylation sites, N32, N104 and N173, was important for α1β2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering β2 subunits. We found that co-expression of γ2 subunits hindered processing of β2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2^+/?) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal β2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the β2 subunit glycosylation sites. The β2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with β2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the β2 subunit N104 N-glycans were involved in altering the function of surface GABA_A receptors.     

The synthesis of 2-acetamido-2-deoxy-6-O-β-d-mannopyranosyl-d-glucose

4,6-Di-O-acetyl-2,3-O-carbonyl-α-d-mannopyranosyl bromide was condensed with benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-α-d-glucopyranoside in the presence of silver carbonate to give crystalline benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-(4,6-di-O-acetyl-2,3-O-carbonyl-β-d-mannopyranosyl)-α-d-glucopyranoside in 32% yield. Removal of the protective O-acetyl and cyclic carbonate groups gave the crystalline benzyl α-glycoside of the disaccharide, which was catalytically hydrogenolyzed to yield the crystalline, title compound. Proof of the anomeric configuration of the interglycosidic linkage was obtained by comparison of the physical, spectral, and chromatographic properties of the disaccharide and its derivatives with those of the previously prepared α-d-linked analog.     

TGF-β2 antagonizes IL-6-promoted cell survival

Molecular and Cellular Biochemistry - Hepatocellular carcinoma (HCC) is one of leading causes of cancer-related death, and its increasing incidence worldwide is a cause for concern. The recombinant...     

Opioid receptor antagonist affinity ligands: 6β-bromoacetamido-6-desoxynaltrexone and 6β-thioglycolamido-6-desoxynaltrexone

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [³H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had K_i values of 1×10^–9 and 1×10^–10 M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5×10^–8 and 1×10^–8 M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [³H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1×10^–7 M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED₅₀ dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.     

β Subunits of Voltage-Gated Calcium Channels

Calcium channel beta subunits have marked effects on the trafficking and on several of the biophysical properties of all high voltage activated calcium channels. In this article I shall review information on the different genes, on the structure of the beta subunits, and on their differential expression and post-translational modification. Their role in trafficking and assembly of the calcium channel heteromultimer will be described, and I will then review their effects on voltage-dependent and kinetic properties, stressing the differences between palmitoylated beta2a and the other beta subunits. Evidence for effects on calcium channel pharmacology will also be examined. I shall discuss the hypothesis that beta subunits can bind reversibly to calcium channels, and examine their role in the G protein modulation of calcium channels. Finally, I shall describe the consequences of knock-out of different beta subunit genes, and describe evidence for the involvement of beta subunits in disease.     

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

γ-Aminobutyric acid type A (GABA_A) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA_A receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABA_A receptors by glycosylation.     

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer

Human hemoglobin (Hb), which is an α₂β₂ tetramer and binds four O₂ molecules, changes its O₂-affinity from low to high as an increase of bound O₂, that is characterized by ‘cooperativity’. This property is indispensable for its function of O₂ transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O₂-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O₂-binding with no cooperativity, indicating the coexistence of two independent hemes with different O₂-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O₂-binding curve with no cooperativity. Resonance Raman, ¹H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O₂-binding to rHb(αH87G), but it did partially occur with O₂-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O₂-binding, but its absence in the β subunit simply enhances the O₂-affinity of α subunit.     

Synthesis of benzyl 2-acetamido-2-deoxy-3-O-β-d-fucopyranosyl-α-d-galactopyranoside and benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-3-O-β-d-fucopyranosyl-α-d-galactopyranoside

Condensation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside with 2,3,4-tri-O-acetyl-α-d-fucopyranosyl bromide in 1:1 nitromethane-benzene, in the presence of powdered mercuric cyanide, afforded benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4-tri-O-acetyl-β-d-fucopyranosyl)-α-d-galactopyranoside (3). Cleavage of the benzylidene group of 3 with hot, 60% aqueous acetic acid afforded diol 4, which, on deacetylation, furnished the disaccharide 5. Condensation of diol 4 with 2-methyl-(3,4,6-tri-O-acetyl-1,2-di-deoxy-α-d-glucopyrano)-[2,1-d]-2-oxazoline in 1,2-dichloroethane afforded the trisaccharide derivative (7). Deacetylation of 7 with Amberlyst A-26 (OH^?) anion-exchange resin in methanol gave the title trisaccharide (8). The structures of 5 and 8 were confirmed by ¹³C-n.m.r. spectroscopy.     

A Tandem Di-hydrophobic Motif Mediates Clathrin-dependent Endocytosis via Direct Binding to the AP-2 ασ2 Subunits

Select plasma membrane proteins can be marked as cargo for inclusion into clathrin-coated pits by common internalization signals (e.g. YXXΦ, dileucine motifs, NPXY) that serve as universal recognition sites for the AP-2 adaptor complex or other clathrin-associated sorting proteins. However, some surface proteins, such as the Kir2.3 potassium channel, lack canonical signals but are still targeted for clathrin-dependent endocytosis. Here, we explore the mechanism. We found an unusual endocytic signal in Kir2.3 that is based on two consecutive pairs of hydrophobic residues. Characterized by the sequence ΦΦXΦΦ (a tandem di-hydrophobic (TDH) motif, where Φ is a hydrophobic amino acid), the signal shows no resemblance to other endocytic motifs, yet it directly interacts with AP-2 to target the Kir2.3 potassium channel into the endocytic pathway. We found that the tandem di-hydrophobic motif directly binds to the ασ2 subunits of AP-2, interacting within a large hydrophobic cleft that encompasses part of the docking site for di-Leu signals, but includes additional structures. These observations expand the repertoire of clathrin-dependent internalization signals and the ways in which AP-2 can coordinate endocytosis of cargo proteins.     

Structures and Functions of Calcium Channel β Subunits

Calcium channel subunits have profound effects on how ₁ subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with ₁ subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of _1C (absent in _1E) and inhibition by G protein dimer of _1E, absent in _1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of ₁ in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca²⁺ channels to account for ₁ alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature ₁ after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition ₁. In the other model, the chaperoning remains associated with the mature channel and ₁ alone channels would in fact be ₁ channels. Upon co-expression of high levels of the regulated channels would have composition [₁].     

6β-Hydroxy-4-stigmasten-3-one and 6β-hydroxy-4-campesten-3-one

The first naturally occurring 6-hydroxylated Δ⁴-3-oxo steroids with intact sterol side chains have been isolated as a molecular complex from the bark extracts of Melia azedarach L. The complex has been characterized by UV, IR, NMR and MS analyses to consist of 6β-hydroxy-4-stigmasten-3-one and 6β-hydroxy-4-campesten-3-one, and these structures confirmed by partial synthesis.     

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor α1‐PDX (α1‐antitrypsin Portland variant), leading to the cell‐surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen‐activated protein kinase). This outside‐in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal‐adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3‐kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up‐regulation of β1 integrins and to their location in larger focal‐adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.     

Calcium Channel α2δ Subunits: Differential Expression, Function, and Drug Binding

Voltage-activated calcium channels are transmembrane proteins that act as transducers of electrical signals into numerous intracellular activities. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. High-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming alpha1 subunit and auxiliary alpha2delta, beta, and--in some tissues--gamma subunits. Auxiliary subunits support the membrane trafficking of the alpha1 subunit and modulate the kinetic properties of the channel. In particular, the alpha2delta subunit has been shown to modify the biophysical and pharmacological properties of the alpha1 subunit. The alpha2delta subunit is posttranslationally cleaved to form disulfide-linked alpha2 and, delta proteins, both of which are heavily glycosylated. Recently it was shown that at least four genes encode for alpha2delta subunits which are expressed in a tissue-specific manner. Their biophysical properties were characterized in coexpression studies with high- and low-voltage-activated calcium channels. Mutations in the gene encoding alpha2delta-2 have been found to underlie the ducky phenotype. This mouse mutant is a model for absence epilepsy and is characterized by spike wave seizures and cerebellar ataxia. Alpha2delta subunits can also support pharmacological interactions with drugs that are used for the treatment of epilepsy and neuropathic pain.     

Misfolded Amyloid Ion Channels Present Mobile β-Sheet Subunits in Contrast to Conventional Ion Channels

In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations. Our Aβ channels consist of the solid-state NMR-based U-shaped β-strand-turn-β-strand motif. In the simulations we obtain ion-permeable channels whose subunit morphologies and shapes are consistent with electron microscopy/atomic force microscopy. In agreement with imaged channels, the simulations indicate that β-sheet channels break into loosely associated mobile β-sheet subunits. The preferred channel sizes (16- to 24-mer) are compatible with electron microscopy/atomic force microscopy-derived dimensions. Mobile subunits were also observed for β-sheet channels formed by cytolytic PG-1 β-hairpins. The emerging picture from our large-scale simulations is that toxic ion channels formed by β-sheets spontaneously break into loosely interacting dynamic units that associate and dissociate leading to toxic ionic flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting α-helices that robustly prevent ion leakage, rather than hydrogen-bonded β-strands. The simulations suggest why conventional gated channels evolved to consist of interacting α-helices rather than hydrogen-bonded β-strands that tend to break in fluidic bilayers. Nature designs folded channels but not misfolded toxic channels.