Richard C. Foreman

Heteropolymerization of S, I, and Z α1-antitrypsin and liver cirrhosis

The association between Z alpha1-antitrypsin deficiency and juvenile cirrhosis is well-recognized, and there is now convincing evidence that the hepatic inclusions are the result of entangled polymers of mutant Z alpha1-antitrypsin. Four percent of the northern European Caucasian population are heterozygotes for the Z variant, but even more common is S alpha1-antitrypsin, which is found in up to 28% of southern Europeans. The S variant is known to have an increased susceptibility to polymerization, although this is marginal compared with the more conformationally unstable Z variant. There has been speculation that the two may interact to produce cirrhosis, but this has never been demonstrated experimentally. This hypothesis was raised again by the observation reported here of a mixed heterozygote for Z alpha1-antitrypsin and another conformationally unstable variant (I alpha1-antitrypsin; 39Arg-->Cys) identified in a 34-year-old man with cirrhosis related to alpha1-antitrypsin deficiency. The conformational stability of the I variant has been characterized, and we have used fluorescence resonance energy transfer to demonstrate the formation of heteropolymers between S and Z alpha1-antitrypsin. Taken together, these results indicate that not only may mixed variants form heteropolymers, but that this can causally lead to the development of cirrhosis.

alpha 1-Antitrypsin Mmalton (Phe52-deleted) forms loop-sheet polymers in vivo. Evidence for the C sheet mechanism of polymerization.

The Z (Glu342 → Lys) and Siiyama (Ser53 → Phe) deficiency variants of α1-antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization in which the reactive center loop of one molecule is inserted into a β-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of processing in the Xenopus oocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma. The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers in vitro more readily than M, but less readily than Z, antitrypsin. The A β-sheet accommodated a reactive center loop peptide much less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the S to R transition, and the formation of the latent conformation. Comparison of the results with similar findings of short chain polymers associated with the antithrombin variant Rouen VI (Bruce, D., Perry, D., Borg, J.-Y., Carrell, R. W., and Wardell, M. R.(1994) J. Clin. Invest. 94, 2265-2274) suggests that polymerization is more complicated than the mechanism proposed earlier. The Z, Siiyama, and Mmalton mutations favor a conformational change in the antitrypsin molecule to an intermediate between the native and latent forms. This would involve a partial overinsertion of the reactive loop into the A sheet with displacement of strand 1C and consequent loop-C sheet polymerization.

The actions of muscle relaxants at nicotinic acetylcholine receptor isoforms

The pharmacological diversity of the different isoforms of the nicotinic acetylcholine receptor arises from the diversity of the subunits that assemble to form the native receptors. The aim of this study was to investigate the actions of the muscle relaxants d-tubocurarine, pancuronium and vecuronium on different isoforms of nicotinic acetylcholine receptors (mouse foetal muscle, mouse adult muscle and a rat neuronal), using the Xenopus oocyte expression system. Oocytes were injected with cRNAs for alpha, beta, gamma, delta subunits (the native foetal muscle subunit combination), or with cRNAs for alpha, beta, epsilon, delta subunits (the native adult muscle subunit combination), or with cRNAs for alpha4beta2 subunits (a putative native neuronal subunit combination). Acetylcholine had a similar potency at all three subunit combinations (EC50 11.6, 17.4 and 19.1 microM, respectively). At all three receptor types, d-tubocurarine and pancuronium blocked the responses elicited by acetylcholine in a reversible manner. Furthermore, the inhibition of the acetylcholine currents for the foetal and adult nicotinic acetylcholine receptor by pancuronium and d-tubocurarine was independent of the holding voltage over the range -100 to -40 mV. In oocytes expressing the foetal muscle nicotinic acetylcholine receptors the inhibition of the current in response to 100 microM acetylcholine by 10 nM d-tubocurarine was 29 +/- 5% (mean +/- S.E.M.; n = 7), and the inhibition by 10 nM pancuronium was 39 +/- 6% (mean +/- S.E.M.; n = 8; P > 0.05 vs. d-tubocurarine). However, in the adult form of the muscle nicotinic acetylcholine receptor, 10 nM d-tubocurarine and 10 nM pancuronium were both more effective at blocking the response to 100 microM acetylcholine compared to the foetal muscle nicotinic acetylcholine receptor, with values of 55 +/- 5% (P < 0.01; n = 12) and 60 +/- 4% (P < 0.001; n = 10), respectively. Thus the developmental switch from the gamma to the epsilon subunit alters the antagonism of the nicotinic acetylcholine receptor for both pancuronium and d-tubocurarine. Vecuronium was more potent than pancuronium. One nM vecuronium reduced the response to 100 microM acetylcholine by 71 +- 6% (n = 10) for foetal and 63 +/- 5% (n = 4) for adult nicotinic acetylcholine receptors. In the alpha4beta2 neuronal nicotinic acetylcholine receptor combination, 10 nM pancuronium was a more effective antagonist of the response to 100 microM acetylcholine (69 +/- 6%, n = 6) than 10 nM d-tubocurarine (30 +/- 5%; n = 6; P < 0.05 compared to pancuronium). This is in contrast to the adult muscle nicotinic acetylcholine receptor, where pancuronium and d-tubocurarine were equieffective. The expression of the beta2 subunit with muscle alpha, epsilon and delta subunits formed a functional receptor which was blocked by pancuronium and d-tubocurarine in a similar manner to the alphabeta1epsilondelta subunit consistent with the hypothesis that the beta subunit is not a major determinant in the action of this drug at the adult muscle nicotinic acetylcholine receptor.

Enhancement by 5-hydroxytryptamine and analogues of desensitization of neuronal and muscle nicotinic receptors expressed in Xenopus oocytes

1 The action of 5‐hydroxytryptamine (5‐HT) on neuronal and muscle nicotinic acetylcholine receptors (nicotinic AChR) expressed in Xenopus oocytes was studied. 2 5‐HT enhanced the rate of desensitization of the acetylcholine (ACh) current response in all receptor subtypes investigated (muscle, αβ2γδ and α4β2), acting in a dose‐dependent manner. 3 5‐HT also reduced the peak current elicited by ACh in a dose‐dependent manner. The IC50 value for the muscle type receptor was 227 ± 0.44 μm, and 166 ± 0.47 μm and 283 ± 0.28 μm for the combinations αβ2γ and α4β2 respectively. 4 The effect of 5‐HT on the responses to ACh (10 μm) was found to be independent of membrane voltage over the range tested (−80 to −10 mV), and to be readily reversed by washout. 5 The action of 5‐HT could be mimicked by structurally similar molecules. The homologue tryptamine was less potent than 5‐HT in blocking the ACh current, with an IC50 of 1.0 ± 0.02 mM. Ketanserin, a 5‐HT2 receptor antagonist, was more potent than 5‐HT, the IC50 being 49.0 ± 1.4 μm. 6 We postulate that a highly conserved portion of the tertiary structure of nicotinic AChRs, which includes some part of the ACh binding site, has affinity for 5‐HT and structural analogues.

Membrane Clustering and Bungarotoxin Binding by the Nicotinic Acetylcholine Receptor: Role of the β Subunit

Abstract: Nicotinic acetylcholine receptors (nAChRs) are localised at morphologically distinct regions of the postsynaptic membrane by interactions between the receptor subunits and cytoskeletal proteins, such as the 43‐kDa protein. We have used Xenopus oocytes to examine the localisation and pharmacological properties of muscle nAChRs associated with 43‐kDa protein and to compare them with hybrid muscle nAChRs containing a β subunit derived from a neuronal source. Receptors expressed on the oocyte outer membrane were visualised using confocal scanning laser microscopy. Coexpression of mouse muscle subunit α1β1γδ and 43‐kDa protein transcripts produced discrete receptor aggregates with a diameter of 1–5 µm whose function was partially blocked by application of neuronal bungarotoxin (NBT) at 100 nM. Substitution of the β1 subunit by the neuronal β2 protein produced a functioning receptor that did not aggregate in the presence of 43‐kDa protein and was substantially blocked by the same concentration of NBT. Hybrid α1β4γδ receptors exhibited a combination of characteristics in that they clustered like normal muscle subunits in the presence of 43‐kDa protein, but showed a sensitivity to NBT intermediate between that of muscle receptors and that of hybrids containing β2. These results suggest that the β subunit is an important determinant in receptor localisation and sensitivity to NBT.

The effect of amino acid substitutions at position 342 on the secretion of human α1-antitrypsin from Xenopus oocytes

A glutamic acid to lysine change in the Z variant of human α1‐antitrypsin is associated with a failure to secrete the protein from synthesising cells. The block in export of the protein may be caused either by the loss of an acidic residue or the introduction of a basic one at this point in the polypeptide chain. Site‐directed mutagenesis has been used to construct novel α1‐antitrypsin mutants which show that the side chain interactions from Glu‐342 are not obligatory for protein export and it is rather the introduction of a basic residue at this point which produces the intracellular accumulation of the protein.

Residues 1 to 80 of the N-terminal domain of the β subunit confer neuronal bungarotoxin sensitivity and agonist selectivity on neuronal nicotinic receptors

Standard two electrode voltage clamp techniques were used to investigate the response of neuronal nicotinic acetylcholine receptors, expressed in Xenopus oocytes, to various agonists and neuronal bungarotoxin (NBT). The β subunit is an important determinant of the receptors pharmacological profile. Co‐expression of α4 and β2 subunits produced a receptor that was relatively insensitive to cytisine and nicotine and inhibited by NBT, whilst the α4β4 combination produced a receptor that was highly sensitive to cytisine and nicotine but resistant to toxin. The first 80 amino acids of the N‐terminal domain of the β subunit are implicated in these characteristics, since the combination of α4 with a hybrid β subunit comprising amino acids 1 → 80 of β2 and 81 → 416 of β4 became relatively insensitive to nicotine and cytisine and resistant to inhibition by neuronal bungarotoxin.

Characterisation of a light chain loss variant of the BCL1 lymphoma

Vaccination of BALB/c mice with idiotypic (id) IgM derived from the murine B cell lymphoma BCL1, protects the animals from challenge with tumour cells. Escape of the tumour cells from immune control is associated with the selection of variant cells which fail to express significant levels of id IgM on their cell surface. We have previously isolated one such variant, SNAG 1, and shown that, while it expresses less than 10% of the levels of surface IgM of the parental BCL1 lymphoma, it continues to synthesise id material which can be detected within the cell. In this report we present a detailed characterisation of this variant and show that the tumour cells no longer synthesise the lambda light chain. This failure to produce the light chain causes the mu heavy chains in SNAG 1 to remain marooned in the endoplasmic reticulum. The mu heavy chains in SNAG 1 have a normal mol. wt and isoelectric point, and so appear not to be mutated. This is unlike the vast majority of light chain loss variants, in which the heavy chains have been shown to contain deletions. Investigation of the mechanisms responsible for the loss of light chain synthesis demonstrated that, while mRNA for the light chain is present, and of a normal size, there was no production of light chain protein in a cell free system. This indicates that the failure to express light chain by SNAG 1 cells is due to an inability to translate the light chain mRNA into the detectable levels of lambda light chain protein.

Activation of nicotinic acetylcholine receptors expressed in quail fibroblasts: effects on intracellular calcium.

1 The aim of these experiments was to determine the ability of the muscle‐type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium‐sensitive fluorescent dye Indo‐1 was used. 2 Application of the nicotinic agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 ± 22 nM (mean ± s.e.mean; n = 40 cells) rising to 600 ± 81 nM on addition of SDC (10 μm; n=15 cells), whereas no increase in [Ca2+]i was seen in non‐transfected control QT6 fibroblasts (before: 128 ± 9 nM, n = 40; after: 113±13 nM, n= 15). 3 The increase in [Ca2+]i caused by application of SDC was dose‐dependent, with an EC50 value of 12.7 ± 5.9 μm (n=14). 4 The responses to SDC in QF18 cells were blocked by prior application of α‐bungarotoxin (200 nM), by the addition of Ca2+ (100 μm), by removal of Na+ ions from the extracellular solution, or by the voltage‐sensitive calcium channel blockers nifedipine and ω‐conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5 We conclude that activation of the nicotinic AChRs leads to a Na+‐dependent depolarization and hence activation of endogenous voltage‐sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.

Involvement of Na,K-pump in SEPYLRFamide-mediated reduction of cholinosensitivity in Helix neurons

SEPYLRFamide acts as an inhibitory modulator of acetylcholine (ACh) receptors in Helix lucorum neurones. Ouabain, a specific inhibitor of Na,K-pump, (0.1 mM, bath application) decreased the ACh-induced inward current (ACh-current) and increased the leak current. Ouabain decreased the modulatory SEPYLRFamide effect on the ACh-current. There was a correlation between the effects of ouabain on the amplitude of the ACh-current and on the modulatory peptide effect. Ouabain and SEPYLRFamide inhibited the activity of Helix aspersa brain Na,K-ATPase. Activation of Na,K-pump by intracellular injection of 3 M Na acetate or 3 M NaCl reduced the modulatory peptide effect on the ACh-current. An inhibitor of Na/Ca-exchange, benzamil (25 muM, bath application), and an inhibitor of Ca(2+)-pump in the endoplasmic reticulum, thapsigargin (TG, applied intracellularly), both prevented the effect of ouabain on SEPYLRFamide-mediated modulatory effect. Another inhibitor of Ca(2+)-pump in the endoplasmic reticulum, cyclopiazonic acid (applied intracellularly), did not prevent the effect of ouabain on SEPYLRFamide-mediated modulatory effect. These results indicate that Na,K-pump is responsible for the SEPYLRFamide-mediated inhibition of ACh receptors in Helix neurons. Na/Ca-exchange and intracellular Ca(2+) released from internal pools containing TG-sensitive Ca(2+)-pump are involved in the Na,K-pump pathway for the SEPYLRFamide-mediated inhibition of ACh receptors.