scholarly journals Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly.

1990 ◽  
Vol 111 (6) ◽  
pp. 2613-2622 ◽  
Author(s):  
P Blount ◽  
J P Merlie
Keyword(s):  
Acetylcholine Receptor ◽  
Alpha Subunit ◽  
Affinity Binding ◽  
Wild Type ◽  
Nicotinic Acetylcholine ◽  
Rapid Degradation ◽  
High Affinity Binding ◽  
Toxin Binding ◽  
High Affinity ◽  
Delta Subunit

The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin-treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR-expressing cells.

scholarly journals Integrin cytoplasmic domains mediate inside-out signal transduction

1994 ◽  
Vol 124 (6) ◽  
pp. 1047-1059 ◽  
Author(s):  
TE O'Toole ◽  
Y Katagiri ◽  
RJ Faull ◽  
K Peter ◽  
R Tamura ◽  
...  
Keyword(s):  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Alpha Subunit ◽  
Affinity Binding ◽  
Cell Type ◽  
Cytoplasmic Domains ◽  
High Affinity Binding ◽  
High Affinity ◽  
Energy Dependent ◽  
Inside Out

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the .beta. and .delta. chains

Biochemistry ◽  
1987 ◽  
Vol 26 (9) ◽  
pp. 2410-2418 ◽  
Author(s):  
Jerome Giraudat ◽  
Michael Dennis ◽  
Thierry Heidmann ◽  
Pierre Yves Haumont ◽  
Florence Lederer ◽  
...  
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Noncompetitive Blockers

scholarly journals Genetic variants of C2 muscle cells that are defective in synthesis of the alpha-subunit of the acetylcholine receptor.

1987 ◽  
Vol 105 (3) ◽  
pp. 1329-1336 ◽  
Author(s):  
R Black ◽  
D Goldman ◽  
S Hochschwender ◽  
J Lindstrom ◽  
Z W Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Genetic Variants ◽  
Muscle Cells ◽  
Binding Activity ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Wild Type ◽  
S1 Nuclease ◽  
Toxin Binding ◽  
Alpha Bungarotoxin

We have analyzed two genetic variants of C2 muscle cells that have reduced levels of binding activity for alpha-bungarotoxin and have found that both synthesize only low levels of the alpha-subunit of the acetylcholine receptor. In both variants the uptake of 22Na in response to carbachol is diminished in proportion to the reduction in toxin-binding activity. In addition, the kinetic and sedimentation properties of the residual toxin-binding activity in both is indistinguishable from that seen in wild-type cells. Immunoblotting experiments on extracts of the variants using subunit-specific antibodies to alpha- and beta-subunits of the acetylcholine receptor demonstrated that the beta-subunit was present, but failed to detect alpha-subunit. In both variants, the amount of alpha-subunit accumulated after a 5-min period of labeling with [35S]methionine was reduced by over 90%, leading to the conclusion that the alpha-subunit is synthesized at greatly reduced rates. Northern blot and S1 nuclease analysis showed no differences between the alpha-subunit mRNA in wild-type and variant cells.

scholarly journals Activation of Drosophila Sodium Channels Promotes Modification by Deltamethrin

2000 ◽  
Vol 115 (3) ◽  
pp. 305-318 ◽  
Author(s):  
Horia Vais ◽  
Martin S. Williamson ◽  
Susannah J. Goodson ◽  
Alan L. Devonshire ◽  
Jeffrey W. Warmke ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Binding Sites ◽  
Voltage Dependence ◽  
Affinity Binding ◽  
Na Channels ◽  
Wild Type ◽  
High Affinity Binding ◽  
High Affinity ◽  
Channel Opening ◽  
Anemonia Sulcata

kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4–S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by <10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action.

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