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Biomedical subjects

W N Green

Publications and source records attributed to W N Green.

At least 19 recordsLinked to original sources
PubMed2000

Neuronal alpha-bungarotoxin receptors are alpha7 subunit homomers.

Nicotinic acetylcholine receptors in the nervous system are heterogeneous with distinct pharmacological and functional properties resulting from differences in post-translational processing and subunit composition. Because of nicotinic receptor diversity, receptor purification and biochemical characterization have been difficult, and the precise subunit composition of each receptor subtype is poorly characterized. Evidence is presented that alpha-bungarotoxin (Bgt)-binding nicotinic receptors found in pheochromocytoma 12 (PC12) cells are pentamers composed solely of alpha7 subunits. Metabolically labeled, affinity-purified Bgt receptors (BgtRs) consisted of a single 55 kDa band on SDS gels, which was recognized by anti-alpha7 antibodies on immunoblots. Isoelectric focusing separated the 55 kDa band into multiple spots, all recognized by anti-alpha7 antibodies and, therefore, each a differentially processed alpha7 subunit. Cell-surface BgtR subunits, cross-linked to each other and (125)I-Bgt, migrated on gels as a ladder of five bands with each band a multiple of an alpha7 subunit monomer. Similar characteristics of BgtRs from rat brain suggest that they, like PC12 BgtRs, are alpha7 pentamers containing differentially processed alpha7 subunits.

Animals↗
PubMed2000

The unusual nature of epibatidine responses at the alpha4beta2 nicotinic acetylcholine receptor.

The identification of an equatorial frog toxin, epibatidine, as a potent non-morphinic analgesic, selective for neuronal nicotinic acetylcholine receptors, provoked a marked renewal in our understanding of pain and its mechanisms. In this work we have examined the effects of epibatidine at the major brain rat alpha4beta2 nicotinic acetylcholine receptor expressed in a cell line. Fast drug applications obtained with a modified liquid filament system were used for the analyses of the currents evoked by acetylcholine, nicotine and epibatidine. Characterized by a slow onset and offset, epibatidine responses were of smaller amplitude to those evoked by acetylcholine or nicotine. About a thousand times more sensitive to epibatidine than acetylcholine, the alpha4beta2 receptor also displayed a more pronounced apparent desensitization to this compound. Finally, overnight exposure to 1 nM epibatidine failed to produce the functional upregulation observed with nicotine. These data indicate that, at the rat alpha4beta2 receptor, epibatidine acts as a partial agonist causing a pronounced inhibition of agonist evoked currents at concentrations that do not activate the receptors.

Algorithms↗
PubMed1999

Nicotinic receptor assembly requires multiple regions throughout the gamma subunit.

Assembly of ionotropic neurotransmitter receptors typified by acetylcholine receptors (AChRs) is thought to be directed by an N-terminal extracellular domain of a subunit. Consistent with this hypothesis, chimeras with the delta subunit N-terminal domain fused to the rest of the gamma subunit can substitute for delta, but not gamma, subunits during AChR assembly. However, chimeras with the gamma subunit N-terminal domain fused to the rest of the delta subunit cannot substitute for gamma or delta subunits during assembly. Furthermore, expression of this chimera with the four wild-type subunits prevents the formation of alpha-bungarotoxin (Bgt) binding sites. Instead of AChR pentamers, complexes are assembled containing only the chimera and either alpha or beta subunits. Based on the results of additional gamma-delta chimeras, there are at least two different regions within the C-terminal half of the chimera required for the dominant-negative effect. Our results indicate that the N-terminal domain of the gamma subunit mediates the initial subunit associations, whereas signals in the C-terminal half of the subunit are required for subsequent subunit interactions.

Animals↗
PubMed1999

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.

Amino Acid Sequence↗
PubMed1998

Formation of the nicotinic acetylcholine receptor binding sites.

Nicotinic acetylcholine receptors (AChRs) are activated by ACh binding to two sites located on different alpha subunits. The two alpha subunits, alpha gamma and alpha delta, are distinguished by their interface with gamma and delta subunits. We have characterized the formation of the ACh binding sites and found, contrary to the current model, that the sites form at different times and in a set order. The first site forms on alpha gamma subunits during the process of subunit assembly. Our data are consistent with the appearance of this site on alpha beta gamma delta subunit tetramers soon after the site for the competitive antagonist alpha-bungarotoxin has formed and delta subunits have assembled with alpha beta gamma trimers. The second site is located on alpha delta subunits and forms after AChR subunits have assembled into alpha2 beta gamma delta pentamers. By determining the order in which the ACh binding sites form, we have also identified the sites in which the delta and second alpha subunits associate during subunit assembly.

Animals↗
PubMed1998

Transient expression of heteromeric ion channels.

Transient transfection is an excellent method for the expression and study of cell-surface, heteromeric ion channels. The cell type, the total amount of DNA, the combination of subunits and the ratio of subunit DNA are all important parameters to consider when attempting to optimize expression. A serious drawback of this method is that the efficiency of subunit assembly is very low in comparison to the efficiency of assembly for stably expressed heteromeric ion channels. The low efficiency of assembly prevents use of transient expression methods for detailed studies of heteromeric AChR assembly, and caution should be taken in the use of these methods for the study of intracellular heteromeric ion channel subunits. After the transient expression of heteromeric AChR subunits, virtually all of the expressing cells contained all four AChR subunits. However, the subunits were heterogeneously distributed among the cells, and the low efficiency of AChR assembly appears to be due to cell-to-cell variations in the ratio of the four subunits.

Animals↗
PubMed1997

Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors.

We have characterized the alpha-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of alpha7-containing receptors that bind alpha-Bgt with high affinity (Kd = 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and alpha-Bgt block approximately 90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3-2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric alpha7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for alpha7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and alpha7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and alpha7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Acetylcholine↗
PubMed1997

The role of the cystine loop in acetylcholine receptor assembly.

Nicotinic acetylcholine receptors (AChRs) are composed of alpha, beta, gamma, and delta subunits, assembled into alpha2betagammadelta pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mM dithiothreitol, AChR subunits assemble into alphabetagamma trimers, but the subsequent steps in assembly are blocked. When alpha subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when beta subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of alphabetagammadelta tetramers and before the addition of the second alpha subunit. After formation of the cystine loop, the alpha subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of alphabetagamma trimers and before the addition of the delta subunit. The data indicate that the alpha subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.

Amino Acid Sequence↗
PubMed1995

Ion-channel assembly.

Transmembrane ion channels regulate the movement of ions (particularly Na+, K+, Ca2+ and Cl-) across cellular membranes, and are critical to numerous aspects of neurobiology. Cells express a diverse array of ion-channel proteins that vary widely in their ion selectivity and in their modulation by ligands (such as neurotransmitters) or by membrane voltage. Most ion channels are multisubunit proteins and, as such, undergo an intricate series of post-translational folding, modification and oligomerization events to achieve their correct functional quaternary structure. The means by which the cell is able to accomplish this complex process of ion-channel assembly is a topic that is beginning to be addressed experimentally.

Calcium↗
PubMed1994

Cyclic AMP-regulated AChR assembly is independent of AChR subunit phosphorylation by PKA.

Forskolin treatment of cells expressing Torpedo acetylcholine receptors leads to enhanced assembly efficiency of subunits, which correlates with increased phosphorylation of the gamma subunit. To determine the role of the two potential protein kinase A sites of the gamma subunit in receptor assembly, cell lines expressing different mutant receptors were established. Mouse fibroblast cell lines stably expressing wild-type Torpedo acetylcholine receptor alpha, beta, delta subunits plus one of three gamma subunit mutations (S353A, S354A, or S353,354A) were established to identify the protein kinase A phosphorylation sites of gamma in vivo, and to determine if increased phosphorylation of the gamma subunit leads to enhanced expression of receptors. We found that both serines (353, 354) in gamma are phosphorylated in vivo by protein kinase A, however, phosphorylation of either or both of these sites does not lead to increased assembly efficiency. We established a cell line expressing alpha, beta, and gamma(S353,354A) subunits only (no delta), and found that the presence of delta (or its phosphorylation) is also not necessary for the observed stimulation by forskolin. alpha beta gamma, alpha gamma, and beta gamma associations were stimulated by forskolin but alpha beta and alpha delta interactions were not. These data imply that the presence of gamma is necessary for forskolin action. We postulate that forskolin may stimulate acetylcholine receptor expression through a cellular protein that is involved in the folding and/or assembly of protein complexes, and that forskolin may regulate the action of such a protein through phosphorylation.

3T3 Cells↗
PubMed1993

Acetylcholine receptor assembly: subunit folding and oligomerization occur sequentially.

The temperature sensitivity of nicotinic acetylcholine receptors (AChRs) from T. californica was used to identify steps in AChR subunit folding and oligomerization. Assembly intermediates were isolated by lowering to an assembly-permissive temperature. The earliest identifiable assembly intermediates, alpha beta gamma trimers, form minutes after subunit synthesis. alpha beta gamma delta tetramers are formed slowly by the addition of delta subunits to trimers, and finally a second alpha subunit is added to form alpha 2 beta gamma delta pentamers. Between these oligomerization steps, subunits fold as monitored by alpha-bungarotoxin-binding site formation, appearance of antigenic epitopes, changes in apparent molecular weight, and changes in detergent solubility. Subunit folding requires specific combinations of subunits and correlates in time with subunit additions, suggesting that these subunit folding events contribute to subunit recognition site formation during assembly.

Animals↗
PubMed1991

cAMP stimulation of acetylcholine receptor expression is mediated through posttranslational mechanisms.

When the four Torpedo acetylcholine receptor (AcChoR) subunit cDNAs are stably integrated into the genome of mouse fibroblast cells, alpha 2 beta gamma delta pentamers with proper pharmacological and electrophysiological properties are expressed on the cell surface. Expression of these AcChoRs can be regulated by agents that stimulate intracellular cAMP levels with the result that increased numbers of cell-surface AcChoRs are produced. Theophylline, 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate, cholera toxin, and forskolin stimulated AcChoR cell-surface expression 1.2-, 1.6-, 2.2-, and 2.3-fold, respectively. cAMP-stimulated expression is mediated through a posttranslational mechanism, and the observed increase in surface AcChoRs correlates with increased lifetimes of each newly synthesized subunit. Increased subunit lifetimes are not observed in cell lines expressing each subunit individually, indicating that subunit stabilization arises through heterologous subunit-subunit interactions.

Animals↗
PubMed1991

Acetylcholine receptor assembly is stimulated by phosphorylation of its gamma subunit.

Different combinations of Torpedo acetylcholine receptor (AChR) subunits stably expressed in mouse fibroblasts were used to establish a role for phosphorylation in AChR biogenesis. When cell lines expressing fully functional AChR complexes (alpha 2 beta gamma delta) were labeled with 32P, only gamma and delta subunits were phosphorylated. Forskolin, which causes a 2- to 3-fold increase in AChR expression by stimulating subunit assembly, increased unassembled gamma phosphorylation, but had little effect on unassembled delta. The forskolin effect on subunit phosphorylation was rapid, significantly preceding its effect on expression. The pivotal role of the gamma subunit was established by treating alpha beta gamma and alpha beta delta cell lines with forskolin and observing increased expression of only alpha beta gamma complexes. This effect was also observed in alpha gamma, but not alpha delta cells. We conclude that the cAMP-induced increase in expression of cell surface AChRs is due to phosphorylation of unassembled gamma subunits, which leads to increased efficiency of assembly of all four subunits.

Animals↗
PubMed1991

Efficiency of acetylcholine receptor subunit assembly and its regulation by cAMP.

Assembly of nicotinic acetylcholine receptor (AChR) subunits was investigated using mouse fibroblast cell lines stably expressing either Torpedo (All-11) or mouse (AM-4) alpha, beta, gamma, and delta AChR subunits. Both cell lines produce fully functional cell surface AChRs. We find that two independent treatments, lower temperature and increased intracellular cAMP can increase AChR expression by increasing the efficiency of subunit assembly. Previously, we showed that the rate of degradation of individual subunits was decreased as the temperature was lowered and that Torpedo AChR expression was acutely temperature sensitive, requiring temperatures lower than 37 degrees C. We find that Torpedo AChR assembly efficiency increases 56-fold as the temperature is decreased from 37 to 20 degrees C. To determine how much of this is a temperature effect on degradation, mouse AChR assembly efficiencies were determined and found to be only approximately fourfold more efficient at 20 than at 37 degrees C. With reduced temperatures, we can achieve assembly efficiencies of Torpedo AChR in fibroblasts of 20-35%. Mouse AChR in muscle cells is also approximately 30% and we obtain approximately 30% assembly efficiency of mouse AChR in fibroblasts (with reduced temperatures, this value approaches 100%). Forskolin, an agent which increases intracellular cAMP levels, increased subunit assembly efficiencies twofold with a corresponding increase in cell surface AChR. Pulse-chase experiments and immunofluorescence microscopy indicate that oligomer assembly occurs in the ER and that AChR oligomers remain in the ER until released to the cell surface. Once released, AChRs move rapidly through the Golgi membrane to the plasma membrane. Forskolin does not alter the intracellular distribution of AChR. Our results indicate that cell surface expression of AChR can be regulated at the level of subunit assembly and suggest a mechanism for the cAMP-induced increase in AChR expression.

Acetylglucosaminidase↗
PubMed1991

Analysis of early events in acetylcholine receptor assembly.

Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into alpha 2 beta gamma delta pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains (Paulson, H.L., and T. Claudio. 1990. J. Cell Biol. 110:1705-1717). We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of alpha, beta, gamma, and delta subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of alpha, contain some disulfide-linked subunits. The coprecipitation of alpha subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associated with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. This fraction increases at temperatures more permissive for assembly, further indicating the importance of the monomer as the precursor to assembly of alpha 2 beta gamma delta pentamers.

Animals↗
PubMed1991

Steady-state gating of batrachotoxin-modified sodium channels. Variability and electrolyte-dependent modulation.

The steady-state gating of individual batrachotoxin-modified sodium channels in neutral phospholipid bilayers exhibits spontaneous, reversible changes in channel activation, such that the midpoint potential (Va) for the gating curves may change, by 30 mV or more, with or without a change in the apparent gating valence (za). Consequently, estimates for Va and, in particular, za from ensemble-averaged gating curves differ from the average values for Va and za from single-channel gating curves. In addition to these spontaneous variations, the average Va shifts systematically as a function of [NaCl] (being -109, -88, and -75 mV at 0.1, 0.5, and 1.0 M NaCl), with no systematic variation in the average za (approximately 3.7). The [NaCl]-dependent shifts in Va were interpreted in terms of screening of fixed charges near the channels' gating machinery. Estimates for the extracellular and intracellular apparent charge densities (sigma e = -0.7 and sigma i = -0.08 e/nm2) were obtained from experiments in symmetrical and asymmetrical NaCl solutions using the Gouy-Chapman theory. In 0.1 M NaCl the extracellular and intracellular surface potentials are estimated to be -94 and -17 mV, respectively. The intrinsic midpoint potential, corrected for the surface potentials, is thus about -30 mV, and the standard free energy of activation is approximately -12 kJ/mol. In symmetrical 0.1 M NaCl, addition of 0.005 M Ba2+ to the extracellular solution produced a 17-mV depolarizing shift in Va and a slight reduction in za. The shift is consistent with predictions using the Gouy-Chapman theory and the above estimate for sigma e. Subsequent addition of 0.005 M Ba2+ to the intracellular solution produced a approximately 5-mV hyperpolarizing shift in the ensemble-averaged gating curve and reduced za by approximately 1. This Ba(2+)-induced shift is threefold larger than predicted, which together with the reduction in za implies that Ba2+ may bind at the intracellular channel surface.

Batrachotoxins↗