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Biomedical subjects

W N Green

Publications and source records attributed to W N Green.

12 recordsLinked to original sources
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↗
PubMed1990

Synaptic contact between embryonic neurons and acetylcholine receptor-fibroblast.

1. Mouse fibroblast cell lines were established that stably express Torpedo californica acetylcholine receptors (AChR) on their cell surface in quantities sufficient for biochemical and pharmacological analyses, as well as electrophysiological analysis at the single channel level. 2. Surface-expressed AChRs were shown to be assembled into proper alpha 2 beta gamma delta pentamers. 3. The distribution of surface-AChRs was uniform and identical in every cell. 4. We were able to successfully coculture AChR-fibroblasts with 1-day old Xenopus laevis embryonal neurons and maintain expression of cell surface AChRs. 5. Using the voltage-clamp technique, miniature end-plate currents were recorded from AChR-fibroblasts which were contacted by neurons. The current amplitudes of these AChRs were approximately 10-fold smaller than those observed in Xenopus myocytes, and the rise-times were slower.

Action Potentials↗
PubMed1989

Fibroblasts transfected with Torpedo acetylcholine receptor beta-, gamma-, and delta-subunit cDNAs express functional receptors when infected with a retroviral alpha recombinant.

Torpedo californica acetylcholine receptor (AChR) alpha-, beta-, gamma-, and delta-subunit cDNAs were each stably introduced into muscle and/or fibroblast cell lines using recombinant retroviral vectors and viral infection, or using SV-40 vectors and DNA-mediated cotransfection. The expressed proteins were characterized in terms of their molecular mass, antigenicity, posttranslational processing, cell surface expression, stability in fibroblasts, stability in differentiated and undifferentiated muscle cells, and ability (of alpha) to bind alpha-bungarotoxin (BuTx). We demonstrated that the alpha, beta, gamma, and delta polypeptides acquired one, one, two, and three units of oligosaccharide, respectively. If all four subunits were expressed in the same cell, fully functional cell surface AChRs were produced which had a Kd for BuTx of 7.8 X 10(-11) M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the Kd for BuTx binding to individual alpha polypeptides was only approximately 4 X 10(-7) M. The half-lives of the alpha, gamma, and delta subunits at 37 degrees C were all found to be quite short (approximately 43 min), while the half-life of the beta subunit was found to be even shorter (approximately 12 min). The unique half-life of the beta subunit suggests that it might perform a key regulatory role in the process of AChR subunit assembly. One stable fibroblast cell line was established by transfection that expressed beta, gamma, and delta subunits simultaneously. When this cell line was infected with a retroviral alpha recombinant, fully functional cell surface AChRs were produced. The successful expression of this pentameric protein complex combining transfection and infection techniques demonstrates one strategy for stably introducing the genes of a heterologous multisubunit protein complex into cells.

Animals↗
PubMed1987

Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts.

Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.

Animals↗
PubMed1987

Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block.

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.

Animals↗
PubMed1987

Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures.

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.

Animals↗