Functional studies of threonine 310 mutations in Glut1: T310I is pathogenic, causing Glut1 deficiency

We have previously reported on a patient with the Glut1 deficiency syndrome (Online Mendelian Inheritance in Man number 606777) carrying a heterozygous T310I missense mutation in the GLUT1 gene (Klepper, J., Wang, D., Fischbarg, J., Vera, J. C., Jarjour, I. T., O'Driscoll, K. R., and De Vivo, D. C. (1999) Neurochem. Res. 24, 587-594). To investigate the molecular basis for the associated functional deficit, we constructed T310A, T310S, and T310I human GLUT1 mutants for expression in Xenopus laevis oocytes via cRNA injection. For all mutants, glucose transport was decreased, and osmotic water permeability (Pf) was increased. Km values for 3-O-methylglucose (3-OMG) uptake under zero-trans influx and equilibrium exchange influx conditions were, respectively, 13 +/- 1 and 68 +/- 5 mm for wild-type Glut1, 5 +/- 1 and 25 +/- 6 mm for T310A, 6 +/- 3 and 30 +/- 6 mm for T310I, and 5 +/- 1 and 48 +/- 5 mm for T310S. Compared with wild-type Glut1, we determined the following. (a). Zero-trans and equilibrium exchange influx values of 3-OMG were significantly decreased, respectively, 15 and 5% in T310A, 8 and 3% in T310I, and 40 and 34% in T310S mutants. (b). Zero-trans efflux of 3-OMG and dehydroascorbic acid uptake were significantly decreased in mutants. (c). The relative Pf values for T310A, T310I, and T310S were increased 3-, 4.8-, and 3.5-fold compared with wild-type values. We found a very high negative correlation between the rate of glucose uptake and Pf (-0.93), and between hydropathy and uptake (-0.92), a moderate correlation between hydropathy and Pf (0.73), and a minimal correlation between uptake, Pf, and molecular weight. These findings are consistent with a central role for hydropathy rather than size at position 310 of this mutation.     

Effects of selective hyperglycemia and hyperinsulinemia on glucose transporters in fetal ovine skeletal muscle

We measured net fetal glucose uptake rate from the placenta, shown previously to be equal to total fetal glucose utilization rate (GUR(f)) and proportional to fetal hindlimb skeletal muscle glucose utilization, under normal conditions and after 1, 2.5, and 24 h of selective hyperglycemia increasing G or selective hyperinsulinemia increasing I. We simultaneously measured the amount of Glut 1 and Glut 4 glucose transporter proteins in fetal sheep skeletal muscle. With increasing G , GUR(f) was increased approximately 40% at 1 and 2.5 h but returned to the control rate by 24 h. This transient increasing G -specific increasing GUR(f) was associated with increased plasma membrane-associated Glut 1 (4-fold) and intracellular Glut 4 (3-fold) protein beginning at 1 h. With increasing I, GUR(f) was increased approximately 70% at 1, 2.5, and 24 h. This more sustained increasing I-specific increasing GUR(f) was associated with a significant increase in Glut 4 protein (2-fold) at 2.5 h but no change in Glut 1 protein. These results show that increasing G and increasing I have independent effects on the amount of Glut 1 and Glut 4 glucose transporter proteins in ovine fetal skeletal muscle. These effects are time dependent and isoform specific and may contribute to increased glucose utilization in fetal skeletal muscle. The lack of a sustained temporal correlation between the increase in transporter proteins and glucose utilization rates indicates that subcellular localization and activity of a transporter or tissues other than the skeletal muscle contribute to net GUR(f).     

Role of glucose carrier in human erythrocyte water permeability.

Although the transport properties of human erythrocyte water channels have been well characterized, the identity of the protein(s) mediating water flow remains unclear. Recent evidence that glucose carriers can conduct water raised the possibility that the glucose carrier, which is abundant in human erythrocytes, is the water channel. To test this possibility, water permeabilities and glucose fluxes were measured in large unilamellar vesicles (LUV) containing human erythrocyte lipid alone (lipid LUV), reconstituted purified human erythrocyte glucose carrier (Glut1 LUV), or reconstituted glucose carrier in the presence of other human erythrocyte ghost proteins (ghost LUV). In glucose and ghost LUV, glucose carriers were present at 25% of the density of native erythrocytes, were oriented randomly in the bilayer, and exhibited characteristic inhibition of glucose flux when exposed to cytochalasin B. Osmotic water permeability (Pf, in centimeters per second; n = 4) averaged 0.0012 +/- 0.00033 in lipid LUV, 0.0032 +/- 0.0015 in Glut1 LUV, and 0.006 +/- 0.0014 in ghost LUV. Activation energies of water flow for the three preparations ranged between 10 and 13 kcal/mol; p-(chloromercuri)benzenesulfonate (pCMBS), an organic mercurial inhibitor of erythrocyte water channels, and cytochalasin B did not alter Pf. These results indicate that reconstitution of glucose carriers at high density increases water permeability but does not result in water channel activity. However, because the turnover number of reconstituted carriers is reduced from that of native carriers, experiments were also performed on erythrocyte ghosts with intact water channel function. In ghosts, Pf averaged 0.038 +/- 0.013 (n = 9), while the activation energy for water flow averaged 3.0 +/- 0.3 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colon water transport in transgenic mice lacking aquaporin-4 water channels

Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (-/-) mice. The transepithelial osmotic water permeability coefficient (P(f)) of in vivo perfused colon of +/+ mice, measured using the volume marker (14)C-labeled polyethylene glycol, was 0.016 +/- 0.002 cm/s. P(f) of proximal colon was greater than that of distal colon (0.020 +/- 0.004 vs. 0. 009 +/- 0.003 cm/s, P < 0.01). P(f) was significantly lower in -/- mice when measured in full-length colon (0.009 +/- 0.002 cm/s, P < 0. 05) and proximal colon (0.013 +/- 0.002 cm/s, P < 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. -/- mice (0.80 +/- 0.01 vs. 0.81 +/- 0.01), but there was a slightly higher water content in defecated stool from -/- mice (0.68 +/- 0.01 vs. 0.65 +/- 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 +/- 9 vs. 51 +/- 8 microl. min(-1). g(-1)). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration.     

Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in beta-cells

Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in beta-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 +/- 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 +/- 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 +/- 311; WT, 425 +/- 231 pg x islet(-1) x 35 min(-1); P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 +/- 0.8 mM glucose, compared with 11.8 +/- 0.7 mM in WT (P < 0.05). Older Tg mice secreted less insulin in response to 20 mM glucose (Tg, 2,256 +/- 342; WT, 3,493 +/- 367 pg x islet(-1) x 35 min(-1); P < 0.05). Secretion in response to carbachol was similar in WT and Tg at both ages. Glucose oxidation was blunted in older Tg islets. At 5 mM glucose, islet CO2 production was comparable between Tg and WT. However, WT mice increased islet CO2 production 2.7 +/- 0.4-fold in 20 mM glucose, compared with only 1.4 +/- 0.1-fold in Tg (P < 0.02). Results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, acting at least in part by modulating glucose oxidation pathways. Prolonged excess hexosamine flux results in glucose desensitization and mimics glucose toxicity.     

Trivalent arsenicals and glucose use different translocation pathways in mammalian GLUT1

Rat glucose transporter isoform 1 or rGLUT1, which is expressed in neonatal heart and the epithelial cells that form the blood-brain barrier, facilitates uptake of the trivalent arsenicals arsenite as As(OH)? and methylarsenite as CH?As(OH)?. GLUT1 may be the major pathway for arsenic uptake into heart and brain, where the metalloid causes cardiotoxicity and neurotoxicity. In this paper, we compare the translocation properties of GLUT1 for trivalent methylarsenite and glucose. Substitution of Ser(66), Arg(126) and Thr(310), residues critical for glucose uptake, led to decreased uptake of glucose but increased uptake of CH?As(OH)?. The K(m) for uptake of CH?As(OH)? of three identified mutants, S66F, R126K and T310I, were decreased 4-10 fold compared to native GLUT1. The osmotic water permeability coefficient (P(f)) of GLUT1 and the three clinical isolates increased in parallel with the rate of CH?As(OH)? uptake. GLUT1 inhibitors Hg(II), cytochalasin B and forskolin reduced uptake of glucose but not CH?As(OH)?. These results indicate that CH?As(OH)? and water use a common translocation pathway in GLUT1 that is different to that of glucose transport.     

Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.     

Improved catalytic efficiency and active site modification of 1,4-beta-D-glucan glucohydrolase A from Thermotoga neapolitana by directed evolution

Thermotoga neapolitana 1,4-beta-d-glucan glucohydrolase A preferentially hydrolyzes cello-oligomers, such as cellotetraose, releasing single glucose moieties from the reducing end of the cello-oligosaccharide chain. Using directed evolution techniques of error-prone PCR and mutant library screening, a variant glucan glucohydrolase has been isolated that hydrolyzes the disaccharide, cellobiose, at a 31% greater rate than its wild type (WT) predecessor. The mutant library, expressed in Escherichia coli, was screened at 85 degrees C for increased hydrolysis of cellobiose, a native substrate rather than a chromogenic analog, using a continuous, thermostable coupled enzyme assay. The V(max) for the mutant was 108 +/- 3 units mg(-1), whereas that of the WT was 75 +/- 2 units mg(-1). The K(m) for both proteins was nearly the same. The k(cat) for the new enzyme increased by 31% and its catalytic efficiency (k(cat)/K(m)) for cellobiose also rose by 31% as compared with the parent. The nucleotide sequence of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition in the most active clone caused an isoleucine to threonine amino acid substitution at position 170. Structural models for I170T and WT proteins were derived by sequence homology with Protein Data Bank code 1BGA from Paenibacillus polymyxa. Analysis of the WT and I170T model structures indicated that the substitution in the mutant enzyme repositioned the conserved catalytic residue Asn-163 and reconfigured entry to the active site.     

A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 +/- 0.10% vs. 4.22 +/- 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 +/- 0.38-fold vs. 4.90 +/- 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 +/- 25% over nonischemic control vs. 234 +/- 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 microM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.     

Sodium valproate inhibits glucose transport and exacerbates Glut1-deficiency in vitro

Anticonvulsant sodium valproate interferes with brain glucose metabolism. The mechanism underlying such metabolic disturbance is unclear. We tested the hypothesis that sodium valproate interferes with cellular glucose transport with a focus on Glut1 since glucose transport across the blood-brain barrier relies on this transporter. Cell types enriched with Glut1 expression including human erythrocytes, human skin fibroblasts, and rat astrocytes were used to study the effects of sodium valproate on glucose transport. Sodium valproate significantly inhibited Glut1 activity in normal and Glut1-deficient erythrocytes by 20%-30%, causing a corresponding reduction of Vmax of glucose transport. Similarly, in primary astrocytes as well as in normal and Glut1-deficient fibroblasts, sodium valproate inhibited glucose transport by 20%-40% (P < 0.05), accompanied by an up to 60% downregulation of GLUT1 mRNA expression (P < 0.05). In conclusion, sodium valproate inhibits glucose transport and exacerbates Glut1 deficiency in vitro. Our findings imply the importance of prudent use of sodium valproate for patients with compromised Glut1 function.     

The effects of phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin on cellular glucose transport

Glucose is the principal fuel for brain metabolism and its movement across the blood-brain barrier depends on Glut1. Impaired glucose transport to the brain may have deleterious consequences. For example, Glut1 deficiency syndrome (Glut1DS) is the result of heterozygous loss of function Glut1 mutation leading to energy failure of the brain and subsequently, epileptic encephalopathy. To preserve the integrity of the energy supply to the brain in patients with compromised glucose transport function, consumption of compounds with glucose transport inhibiting properties should be avoided. Phenytoin is a widely used anticonvulsant that affects carbohydrate metabolism. In this study, the hypothesis that phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) affect cellular glucose transport was tested. With a focus on Glut1, the effects of phenytoin and HPPH on cellular glucose transport were studied. Glucose uptake assay measuring the zero-trans influx of radioactive-labeled glucose analogues showed that phenytoin and HPPH did not exert immediate effects on erythrocyte Glut1 activity or glucose transport in Hs68 control fibroblasts, Glut1DS primary fibroblasts isolated from two patients, or in rat primary astrocytes. Prolonged exposure to the two compounds could stimulate glucose transport by up to 30-60% over the control level (p <0.05) in Hs68 and Glut1DS fibroblasts as well as in rat astrocytes. The stimulation of glucose transport by HPPH was dose-dependent and accompanied by an up-regulation of GLUT1 mRNA expression (p <0.05). In conclusion, phenytoin and HPPH do not compromise cellular glucose transport. Prolonged exposure to these compounds can modify carbohydrate homeostasis by up-regulating glucose transport in both normal and Glut1DS conditions in vitro.     

Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes

The amounts of the brain type and muscle type glucose transporters (designated Glut 1 and 4, respectively) in 3T3-L1 adipocytes have been determined by quantitative immunoblotting with antibodies against their carboxyl-terminal peptides. There are about 950,000 and 280,000 copies of Glut 1 and 4, respectively, per cell. Insulin caused the translocation of both types of transporters from an intracellular location to the plasma membrane. The insulin-elicited increase in cell surface transporters was assessed by labeling the surface transporters with a newly developed, membrane-impermeant, photoaffinity labeling reagent for glucose transporters. The increases in Glut 1 and 4 averaged 6.5- and 17-fold, respectively, whereas there was a 21-fold in hexose transport. These results indicate that the translocation of Glut 4 could largely account for the insulin effect on transport rate, but only if the intrinsic activity of Glut 4 is much higher than that of Glut 1. The two transporters are colocalized intracellularly: vesicles (average diameter 72 nm) isolated from the intracellular membranes by immunoadsorption with antibodies against Glut 1 contained 95% of the Glut 4 and, conversely, vesicles isolated with antibodies against Glut 4 contained 85% of the Glut 1.     

Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport

Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.     

Monitoring active site alterations upon mutation of yeast pyruvate kinase using 205Tl+ NMR

The interaction of the monovalent cation with wild type (WT) yeast pyruvate kinase (YPK) and with the T298S, T298C, and T298A mutants was investigated by 205Tl+ NMR to monitor possible structural alterations at the active site by Thr-298 mutation. TlNO3 activates WT YPK with a kcat value similar to that obtained with KCl and an apparent Ka of 0.96 +/- 0.07 mm in the presence of Mn2+ and fructose 1,6-bisphosphate. With the three mutants, Tl+ is a better activator than is K+ based on kcat values. Tl+ activation and inhibition of YPK is affected by mutation of the active site Thr-298. The effect of Mn2+ on the 1/T value of 205Tl+1 in the presence of the WT and mutant YPK complexes was determined at 173 MHz (300 MHz, 1H) and 346 MHz (600 MHz, 1H). For each complex studied, 1/pT2p > 1/pT1p and 1/pT1p is frequency-dependent suggesting fast exchange conditions. The values of 1/pT1p differ for each mutant. A correlation time of 0.65 +/- 0.35 ns was estimated for the Mn2+-205Tl+ interaction. The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T1M of YPK-bound 205Tl+. The calculated Tl+-Mn2+ distance for the Thr-298 mutants is decreased by about 1 A from 6.0 +/- 0.2 A observed with WT. The results suggest conformational alterations at the active site of YPK where phosphoryl transfer occurs upon mutation of Thr-298. These conformational changes may, in part, explain the alteration in kcat and kcat/Km,PEP observed with the Thr-298 mutants.     

Activation of Glut1 glucose transporter in response to inhibition of oxidative phosphorylation.

We have previously shown that exposure of Clone 9 cells to hypoxia, cyanide, or azide results in an acute stimulation of glucose transport that is largely mediated by "activation" of glucose transporter (Glut1) sites preexisting in the plasma membrane. However, it is not known whether inhibition of oxidative phosphorylation only at its terminal step, or at any of its steps, leads to the glucose transport response. Hence, the effect of azide (5 mM), rotenone (1 microM), rotenone (1 microM) plus thenoyltrifluoroacetone (TTFA) (5 microM), antimycin A (0.3 microM), dinitrophenol (0.25 mM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (2.5 microM), and oligomycin B (0.15 microM) on glucose transport was determined. All of the above agents elicited a similar approximately 4-fold stimulation of cytochalasin B (CB)-inhibitable 3-O-methyl glucose (3-OMG) uptake in Clone 9 cells. The stimulatory effect of azide on 3-OMG uptake was not inhibited by antioxidants 2-mercaptopropionyl glycine (1.2 mM) and 1,10-phenanthroline (40 microM), while, in contrast, the antioxidants attenuated the stimulation of glucose transport in response to 250 microM H(2)O(2) by approximately 50%. To differentiate between an increase in the number of functional Glut1 sites in the plasma membrane (in the absence of "translocation") versus an increase in the "intrinsic activity" of Glut1, the effect of azide on the energy of activation (E(a)) of glucose transport was measured. The E(a) was determined by measuring the rate of CB-inhibitable 3-OMG uptake at 24.0, 28.0, 35. 0, and 40 degrees C. The E(a) of control Clone 9 cells and of cells exposed to 10 mM azide for 2 h was 32,530 +/- 1830 and 31,220 +/- 600 J/mol, respectively (P > 0.1), while the rate of CB-inhibitable 3-OMG uptake was 9.3 +/- 0.7-fold higher in azide-treated cells. It is concluded that (i) inhibition of oxidative phosphorylation, at any of its steps, leads to a stimulation of glucose transport, and (ii) the mechanism of stimulation of glucose transport in response to azide appears to be predominately mediated by an apparent increase in the number of functional Glut1 sites in the plasma membrane (instead of an increase in their "intrinsic activity"), suggesting an "unmasking" mechanism.     

Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor beta 1 subunit.

The Ca2+ currents, charge movements, and intracellular Ca2+ transients in mouse skeletal muscle cells homozygous for a null mutation in the cchb1 gene encoding the beta 1 subunit of the dihydropyridine receptor have been characterized. I beta null, the L-type Ca2+ current of mutant cells, had a approximately 13-fold lower density than the L-type current of normal cells (0.41 +/- 0.042 pA/pF at + 20 mV, compared with 5.2 +/- 0.38 pA/pF in normal cells). I beta null was sensitive to dihydropyridines and had faster kinetics of activation and slower kinetics of inactivation than the L-type current of normal cells. Charge movement was reduced approximately 2.8-fold, with Qmax = 6.9 +/- 0.61 and Qmax = 2.5 +/- 0.2 nC/microF in normal and mutant cells, respectively. Approximately 40% of Qmax was nifedipine sensitive in both groups. In contrast to normal cells, Ca2+ transients could not be detected in mutant cells at any test potential; however, caffeine induced a robust Ca2+ transient. In homogenates of mutant muscle, the maximum density of [3H]PN200-110 binding sites (Bmax) was reduced approximately 3.9-fold. The results suggest that the excitation-contraction uncoupling of beta 1-null skeletal muscle involves a failure of the transduction mechanism that is due to either a reduced amount of alpha 1S subunits in the membrane or the specific absence of beta 1 from the voltage-sensor complex.     

T cell receptors employ diverse strategies to target a p53 cancer neoantigen

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells.     

Differential accumulation of Glut1 in the non-DRM domain of the plasma membrane in response to the inhibition of oxidative phosphorylation

Approximately 50% of Glut1 in the plasma membrane of Clone 9 cells is localized to the detergent-resistant membrane (DRM) fraction. Acute exposure (90 min) to 5mM azide stimulated glucose transport by approximately 4.7-fold and increased the abundance of Glut1 in the non-DRM fraction of the plasma membrane by approximately 2.9-fold while the abundance of Glut1 in the DRMs was not changed. In parallel experiments, approximately 17 h exposure to azide further increased the rate of glucose transport over that observed at 90 min by approximately 33% and increased plasma membrane Glut1 content by approximately 3.5-fold over control. The increase in total plasma membrane Glut1 reflected a approximately 4.7-fold increase of Glut1 content in the non-DRM fraction and a approximately 2.6-fold increase in the DRMs. We conclude that acute exposure to azide increases Glut1 content in the non-DRM fractions, while prolonged exposure to azide increases the Glut1 content in both non-DRM and DRM fractions. These changes may play an important role in the stimulation of glucose transport in response to the inhibition of oxidative phosphorylation.     

Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for β-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.     

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.