Role of peroxisome proliferator-activated receptor α in disease of pancreatic β cells

Expression of peroxisome proliferator-activated receptor α (PPARα) and enzymes of fatty acid (FA) oxidation is markedly reduced in the fat-laden, dysfunctional islets of obese, prediabetic Zucker diabetic fatty (fa/fa) rats with mutated leptin receptors (OB-R). Leptin, PPARα/retinoid x receptor ligands, and FA all up-regulate PPARα and enzymes of FA oxidation and stimulate [³H]-palmitate oxidation in normal islets but not in islets from fa/fa rats. Overexpression of normal OB-R in islets of fa/fa rats corrects all of the foregoing abnormalities and reverses the diabetic phenotype. PPARα is a OB-R-dependent factor required for normal fat homeostasis in islet cells.     

Fatty acid-induced β cell apoptosis: A link between obesity and diabetes

Like obese humans, Zucker diabetic fatty (ZDF) rats exhibit early β cell compensation for insulin resistance (4-fold β cell hyperplasia) followed by decompensation (>50% loss of β cells). In prediabetic and diabetic ZDF islets, apoptosis measured by DNA laddering is increased 3- and >7-fold, respectively, compared with lean ZDF controls. Ceramide, a fatty acid-containing messenger in cytokine-induced apoptosis, was significantly increased (P < 0.01) in prediabetic and diabetic islets. Free fatty acids (FFAs) in plasma are high (>1 mM) in prediabetic and diabetic ZDF rats; therefore, we cultured prediabetic islets in 1 mM FFA. DNA laddering rose to 19.6% vs. 4.6% in lean control islets, preceded by an 82% increase in ceramide. C₂-Ceramide without FFA induced DNA laddering, but fumonisin B₁, a ceramide synthetase inhibitor, completely blocked FFA-induced DNA laddering in cultured ZDF islets. [³H]Palmitate incorporation in [³H]ceramide in ZDF islets was twice that of controls, but [³H]palmitate oxidation was 77% less. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, both blocked DNA laddering. These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis. In ZDF obesity, β cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production.     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

Gsα-selective G protein antagonists

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the G_α subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for G_sα. Two compounds have been identified: NF449 (4,4′,4",4′"-[carbonyl-bis[imino-5,1,3-benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3-disulfonate) and NF503 (4,4′-[carbonylbis[imino-3,1-phenylene-(2,5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds (i) suppress the association rate of guanosine 5′-[γ-thio]triphosphate ([³⁵S]GTP[γS]) binding to G_sα-s but not to G_iα-1, (ii) inhibit stimulation of adenylyl cyclase activity in S49 cyc⁻ membranes (deficient in endogenous G_sα) by exogenously added G_sα-s, and (iii) block the coupling of β-adrenergic receptors to G_s with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A₁-adenosine receptor with its cognate G proteins (G_i/G_o) at concentrations that are >30-fold higher than those required for uncoupling of β-adrenergic receptor/G_s tandems; similarly, the angiotensin II type-1 receptor (a prototypical G_q-coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for G_sα-selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition.     

Effects of Zn2+ on wild and mutant neuronal α7 nicotinic receptors

Zn²⁺ is a key structural/functional component of many proteins and is present at high concentrations in the brain and retina, where it modulates ligand-gated receptors. Therefore, a study was made of the effects of zinc on homomeric neuronal nicotinic receptors expressed in Xenopus oocytes after injection of cDNAs encoding the chicken wild or mutant α₇ subunits. In oocytes expressing wild-type receptors, Zn²⁺ alone did not elicit appreciable membrane currents. Acetylcholine (AcCho) elicited large currents (I_AcCho) that were reduced by Zn²⁺ in a reversible and dose-dependent manner, with an IC₅₀ of 27 μM and a Hill coefficient of 0.4. The inhibition of I_AcCho by Zn²⁺ was competitive and voltage-independent, a behavior incompatible with a channel blockade mechanism. In sharp contrast, in oocytes expressing a receptor mutant, with a threonine-for-leucine 247 substitution (^L247Tα₇), subnanomolar concentrations of Zn²⁺ elicited membrane currents (I_Zn) that were reversibly inhibited by the nicotinic receptor blockers methyllycaconitine and α-bungarotoxin. Cell-attached single-channel recordings showed that Zn²⁺ opened channels that had a mean open time of 5 ms and a conductance of 48 pS. At millimolar concentrations Zn²⁺ reduced I_AcCho and the block became stronger with cell hyperpolarization. Thus, Zn²⁺ is a reversible blocker of wild-type α₇ receptors, but becomes an agonist, as well as an antagonist, following mutation of the highly conserved leucine residue 247 located in the M2 channel domain. We conclude that Zn²⁺ is a modulator as well as an activator of homomeric nicotinic α₇ receptors.     

Association of calcium channel α1S and β1a subunits is required for the targeting of β1a but not of α1S into skeletal muscle triads

The skeletal muscle L-type Ca²⁺ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca²⁺ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α_1S–β_1a interactions in the incorporation of the functional channel complex into the triad, α_1S and β_1a [or a β_1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α_1S subunit was diffusely distributed in the cytoplasm. On coexpression with the α_1S subunit βGFP distribution became clustered and colocalized with α_1S immunofluorescence. Based on the colocalization of βGFP and α_1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α_1S with and without β_1a restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α_1S mutants in which the β interaction domain had been altered (α_1S-Y366S) or deleted (α_1S-Δ351–380). Although α_1S-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. Thus, β_1a requires the association with the β interaction domain in the I–II cytoplasmic loop of α_1S for its own incorporation into triad junctions, but stable α_1S–β_1a association is not necessary for the targeting of α_1S into the triads or for its normal function in Ca²⁺ conductance and excitation-contraction coupling.     

A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABA_A) receptors. Coexpression of either rat γ₂ or γ₃, in combination with α₁ and β₂ subunits, results both in receptors displaying high [³H]Ro 15-1788 affinity. However, receptors containing a γ₃ subunit display a 178-fold reduced affinity to zolpidem as compared with γ₂-containing receptors. Eight chimeras between γ₂ and γ₃ were constructed followed by nine different point mutations in γ₂, each to the homologous amino acid residue found in γ₃. Chimeric or mutant γ subunits were coexpressed with α₁ and β₂ in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ₂ (γ₂M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [³H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ₂M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ₂M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ₂M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABA_A receptors. As the modulatory site in the GABA_A receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ₂M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.     

Functional expression of a novel human neurokinin-3 receptor homolog that binds [3H]senktide and [125I-MePhe7]neurokinin B, and is responsive to tachykinin peptide agonists

In 1992, Xie et al. identified a cDNA sequence in the expression cloning search for the κ opioid receptor. When the cDNA was expressed in Cos-7 cells, binding of opioid compounds was observed to be of low affinity and without κ, μ, or δ selectivity [Xie, G.-X., Miyajima, A. and Goldstein, A. (1992) Proc. Natl. Acad. Sci. USA 89, 4124–4128]. This cDNA was highly homologous to the human neurokinin-3 (NK-3) receptor sequence, and displayed lower homology to NK-1 and NK-2 sequences. This sequence was stably expressed in Chinese hamster ovary cells, which do not express neurokinin receptors naturally, and ligand binding and second messenger characteristics were compared with a human NK-3 receptor. The NK-3 receptor homolog bound [³H]senktide with a K_d of 39 nM, similar to that of the NK-3 receptor. The rank order of tachykinin peptides competing for [³H]senktide binding at the NK-3 receptor homolog was [MePhe⁷]neurokinin B > senktide > substance P = neurokinin A > neurokinin B. This cell line also bound [¹²⁵I-MePhe⁷]neurokinin B; however, neurokinin B was an effective competitor. Tachykinin peptides stimulated both inositol phospholipid hydrolysis and arachidonic acid release at NK-3 and NK-3 receptor homolog cell lines, with similar rank orders of potency of [MePhe⁷]neurokinin B = neurokinin B = senktide > NKA = substance P. These results indicate that expression of the NK-3 receptor homolog cDNA in the Chinese hamster ovary cell system induces the expression of a receptor site with many similarities but certain key differences from that of the human NK-3 receptor. The results are discussed with reference to the existence of a novel human tachykinin receptor.     

Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers

Streaming potentials across cloned epithelial Na⁺ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na⁺] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na⁺ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.     

Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10⁻⁸ M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using ³⁵S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [³⁵S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent K_d values for [³⁵S]PSK-α binding were found to be 1 × 10⁻⁹ M for the high-affinity type and 1 × 10⁻⁷ M for the low-affinity type, with maximal numbers of binding sites of 1 × 10⁴ sites per cell and 1 × 10⁵ sites per cell, respectively. Competition studies with [³⁵S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.     

Targeted inactivation of αi2 or αi3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Cardiac muscarinic receptors activate an inwardly rectifying K⁺ channel, I_K^+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart G_i2, G_i3, or G_o). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive α subunits to determine which G proteins are required for receptor-mediated regulation of I_K^+Ach in intact cells. The muscarinic agonist carbachol increased I_K^+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking α_o, and in cells lacking the PT-insensitive G protein α_q. In cells with targeted inactivation of α_i2 or α_i3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the α_i2- and α_i3-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate I_K^+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking α_i2 or α_i3. Our results show that expression of two specific PT-sensitive α subunits (α_i2 and α_i3 but not α_o) is required for normal agonist-dependent activation of I_K^+Ach and suggest that both α_i2- and α_i3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of I_K^+Ach or the expression of α_i2 or α_i3.     

The γ134.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase

In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with γ₁z34.5⁻ mutants. The carboxyl-terminal 64 aa of γ₁34.5 protein are homologous to the corresponding domain of MyD116, the murine growth arrest and DNA damage gene 34 (GADD34) protein and the two domains are functionally interchangeable in infected cells. This report shows that (i) the carboxyl terminus of MyD116 interacts with protein phosphatase 1α in yeast, and both MyD116 and γ₁34.5 interact with protein phosphatase 1α in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the α subunit in purified eIF-2 phosphorylated in vitro is specifically dephosphorylated by S10 fractions of wild-type infected cells at a rate 3000 times that of mock-infected cells, whereas the eIF-2α-P phosphatase activity of γ₁34.5⁻ virus infected cells is lower than that of mock-infected cells. The eIF-2α-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2α-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [³²P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, γ₁34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2α to enable continued protein synthesis despite the presence of activated PKR. The GADD34 protein may have a similar function in eukaryotic cells. The proposed mechanism for maintenance of protein synthesis in the face of double-stranded RNA accumulation is different from that described for viruses examined to date.     

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α₁β₁ and α₂β₁ integrins—through induction of mRNAs encoding the α₁ and α₂ subunits. In contrast, VEGF did not induce increased expression of the α₃β₁ integrin, which also has been implicated in collagen binding. Integrin α₁-blocking and α₂-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α₁-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α₁β₁ and α₂β₁ expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α₁-blocking and α₂-blocking Abs. In vivo, a combination of α₁-blocking and α₂-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α₁β₁ and α₂β₁ expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α₁β₁ and α₂β₁ antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca²⁺ ions, acting through Ca²⁺-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca²⁺ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α-toxin, including an α-toxin mutant that forms pores whose permeability is modulated by Zn²⁺. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [γ-³²P]ATP to α-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca²⁺]_free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca²⁺]_free conditions, even when rods were loaded with ATP and the pores were closed by Zn²⁺. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca²⁺ and Ca²⁺-binding proteins, including recoverin.     

Posttranslational modification of Gαo1 generates Gαo3, an abundant G protein in brain

Gα_o, the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Gα_o1 and Gα_o2, derived by alternative splicing of the mRNA. A third Gα_o1-related isoform, Gα_o3, has been purified, representing about 30% of total G_o in brain. Initial studies revealed distinct biochemical properties of Gα_o3 as compared with other Gα_o isoforms. In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated Gα_o1 and Gα_o3, C-terminal peptides showed a difference of +1 Da for Gα_o3. Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of Gα_o3. Gel electrophoretic analysis of recombinant Gα_o3 showed the same mobility as native Gα_o3 but distinct to Gα_o1. The conversion of ³⁴⁶Asn→Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5′-[γ-thio]triphosphate binding. No cDNA coding for Gα_o3 was detected, suggesting an enzymatic deamidation of Gα_o1 by a yet-unidentified activity. Therefore, Gα heterogeneity is generated not only at the DNA or RNA levels, but also at the protein level. The relative amount of Gα_o1 and Gα_o3 differed from cell type to cell type, indicating an additional principle of G protein regulation.     

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [³H]-5-HT-moduline bound specifically to rat brain sections with high affinity (K_d = 0.77 nM and B_max = 0.26 dpm/mm²). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT_1B receptor knock-out mice, the specific binding of [³H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT_1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT_1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT_1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT_1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT_1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT_1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.     

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins αvβ3 and αIIbβ3

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin α_vβ₃ (also known as the vitronectin receptor) or α_IIbβ₃ (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin α_IIbβ₃. Defined β₃ mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing α_vβ₃ and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.     

Unmasking the functions of the chromaffin cell α7 nicotinic receptor by using short pulses of acetylcholine and selective blockers

Methyllycaconitine (MLA), α-conotoxin ImI, and α-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 μM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC₅₀s of 100 nM for MLA and 300 nM for α-conotoxin ImI and α-bungarotoxin. MLA (100 nM), α-conotoxin ImI (1 μM), and α-bungarotoxin (1 μM) halved the entry of ⁴⁵Ca²⁺ stimulated by 5-s pulses of 300 μM ACh applied to incubated cells. These supramaximal concentrations of α₇ nicotinic receptor blockers depressed by 30% (MLA), 25% (α-bungarotoxin), and 50% (α-conotoxin ImI) the inward current generated by 1-s pulses of 100 μM ACh, applied to voltage-clamped chromaffin cells. In Xenopus oocytes expressing rat brain α₇ neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, α-conotoxin ImI, and α-bungarotoxin with IC₅₀s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through α₃β₄ nAChR was unaffected by α-conotoxin ImI and α-bungarotoxin, and weakly blocked by MLA (IC₅₀ = 1 μM). The functions of controlling the electrical activity, the entry of Ca²⁺, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to α₃β₄ nAChR; the present results constitute the first evidence to support a prominent role of α₇ nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.     

Modulation of α1β3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a β-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABA_A α1β3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the β⁺α⁻ interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in β3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [β3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1β3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the β⁺α⁻ interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to β3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the β⁺α⁻ interface for the anesthetic propofol.

While photoswitches have been a popular topic in numerous reviews (1), their application in research is still very rare. Photoswitches are freely diffusible molecules containing a photosensitive moiety which can alternate between two isomeric forms depending on light irradiation at specific wavelengths. These isomeric forms of the photoswitch possess different affinities or efficacies toward their biological target, such that their pharmacological activity can be turned on or off depending on the light wavelength used, thus providing temporal and spatial control. When photoswitches covalently tether to a native or engineered residue at the specific biological target, the resulting conformation would determine the corresponding pharmacological activity. One way the tethered photoswitch can be activated is by positioning the tether in such a way that the ligand moiety can reach its binding site in only one of the photoswitch conformations. Additional major advantages of the tethered photoswitches are high local concentration (the residue cannot diffuse away) and spatial restriction within the biological target. The ligand groups of photoswitches can possess diverse pharmacological activities, acting as agonists, inverse agonists, or antagonists at specific binding sites.The GABA_A receptor is formed by five subunits (usually two α, two β, and one γ or δ) arranged in pseudosymmetry around a central channel, in the following order (counterclockwise, viewed from the extracellular side): γ-β-α-β-α (2). Each interface is named after the subunits that form it, with “+” and “−” designated following the counterclockwise order (a model of the α1β3γ2 GABA_A receptor can be found in SI Appendix, Fig. S1). Each subunit consists of an extracellular domain, attached to a sequence of four transmembrane helices (TMs), with a large intracellular loop inserted between TM3 and TM4. Multiple anesthetic drugs that act through this receptor possess relatively low binding affinities; therefore, precise identification and characterization of their binding sites require the use of techniques like mutagenesis, substituted cysteine modification protection (SCAMP), and photolabeling with photoreactive anesthetic analogs (3). Structures of the GABA_A receptor with bound anesthetic drugs have recently been made available (4), but one unexplored way of obtaining valuable, functional information would be by using appropriately designed tethered photoswitches. In previous studies of GABA_A receptors, the photoswitches used have included diffusible propofol photoswitches (5, 6) and a tethered propofol photoswitch with a very long spacer between the tethering cysteine (located in the extracellular domain) and the propofol moiety (6). Though all three positively modulated GABA_A receptors, none could be used to define the propofol binding site.Multiple studies point to propofol binding cavities being located in the TM interfaces between GABA_A receptor subunits. The observation that specific mutations in TM2 or TM3 of GABA_A β subunits could dramatically decrease propofol potentiation of GABA responses, and even direct activation of the receptor (7–9) led to the development of the β3(N265M) knockin mouse, which showed either a greatly decreased or absent hypnotic effect of propofol, depending on the test used (10). More recent studies have focused on a more complete characterization of binding sites for propofol as well as other intravenous anesthetics. A photoreactive propofol analog labeled three amino acids in the β⁺α⁻ interface (β3M286, α1M236, and α1I239) and one in the α⁺β⁻ interface (β3M227) in α1β3 receptors. Using etomidate and R-mTFD-MPAB [R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid] to inhibit photolabeling established that propofol also binds to the β⁺β⁻ interface, suggesting that propofol shows little selectivity for either interface (11). Another study mutated photolabeled residues (α1M236, β3M227, and their homologs, all located in TM1) to tryptophan (causing steric occupancy of the pocket) and cysteine (to test propofol protection against cysteine-specific labeling) (12). SCAMP studies confirmed that propofol binds to the β⁺α⁻ and α⁺β⁻ interfaces, and also to the γ⁺β⁻ interface; there was no evidence of binding to the α⁺γ⁻ interface. A more recent study has expanded the analysis of residues at the β⁺α⁻ interface that line a putative propofol binding site (13) (SI Appendix, Fig. S1). And cryoelectron microscopy (cryo-EM) structures of α1β2γ2 GABA_A receptors bound to intravenous anesthetics and benzodiazepines have recently been published (4), consolidating the evidence toward a propofol binding site at the β⁺α⁻ interfaces.Our aim was to develop a β-subunit-tethered propofol photoswitch, as a tool to better study the mechanism of anesthesia through the GABA_A α1β3ɣ2 receptor, merging both structural and functional approaches. This tethered photoswitch (Fig. 1A) consists of propofol as the ligand group, linked to an azobenzene group (which is photosensitive and can change between cis and trans isomeric forms, Fig. 1B), and finally a tethering group (methanethiosulfonate, that spontaneously forms a covalent bond with the thiolate group of cysteines located in water-filled cavities). This photoswitch was abbreviated as MAP20 (methanethiosulfonate azobenzene propofol 2020). These three basic components of this tethered photoswitch are connected by short spacers, decreasing the range between tethering and target residues. We used β3 subunits in our study because the immobilizing and hypnotic effects of propofol are mostly mediated by β3-containing receptors (10).Open in a separate windowFig. 1.Tethered photoswitch. (A) Methanethiosulfonate azobenzene propofol 2020 (MAP20) consists of propofol (ligand group), an azobenzene moiety (photosensitive), and a tether (methanethiosulfonate). (B) The azobenzene moiety can change between cis and trans isomeric forms depending on the wavelength of the light irradiated.     

Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α₁ pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α₁ gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca²⁺ concentration [Ca²⁺]_i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K⁺-stimulated [Ca²⁺]_i increase in the α_1A cell line but not in the α_1B, α_1C, α_1D, and α_1E cell lines, establishing the α_1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α_1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs.