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Structural and Functional Studies on the Subunits of the Nicotinic Acetylcholine Receptor

Citation

Mayne, Katherine Mixter (1986) Structural and Functional Studies on the Subunits of the Nicotinic Acetylcholine Receptor. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/nvv4-qt08. https://resolver.caltech.edu/CaltechTHESIS:04152019-153632089

Abstract

An introduction to the work in the study of the nicotinic acetylcholine receptor is presented. The author reviews the field to place the work of the present volume in its proper context The major developments in studying the protein biochemistry of the receptor are reviewed, including the subunit makeup, ligand binding, and protein sequences. These studies led to the cloning and sequencing of many of the subunits as cDNA or genomic DNA constructions. This wealth of sequence information has allowed the formulation of detailed models of receptor structure. Current work centers on testing various aspects of these models and expanding the scope of the field into different species and tissues that utilize this receptor.

Partial cDNA clones specific for the β and δ subunits of the acetylcholine receptor of Torpedo californica were isolated by the following method. A cDNA library was constructed from electric organ poly( A)+ RNA and enriched by screening for clones more abundantly represented in electric organ than in brain or-liver mRNA preparations. These clones were tested by hybridization selection of clone specific mRNA which was then translated in vitro. Protein products were immunoprecipitated and analyzed by gel electrophoresis. The isolated clones were used to screen a library of Torpedo genomic DNA which resulted in the isolation of the gene for the Torpedo δ subunit. The δ gene was found to be single copy in Torpedo, and it contains at least four introns.

A cDNA library was constructed in λgt10 from membrane bound poly(A)+ RNA from mouse BC3H-1 cells. This library was screened with cDNA encoding the complete protein region of the Torpedo γ and δ subunits. Positively hybridizing clones isolated with the Torpedo γ subunit were sequenced and compared with published data. The deduced amino acid sequence was more highly homologous to the Torpedo δ than to the Torpedo γ and on this basis the mouse clone was tentatively identified as a δ subunit of the acetylcholine receptor. The mouse nucleotide sequence has several stretches of strong homology with the Torpedo γ subunit cDNA, but no such homology with the Torpedo δ subunit . A genomic blotting experiment indicated that there is probably one, but at most two chromosomal genes encoding this or closely related sequences.

In order to test the assignment of the mouse δ cDNA by a more functional criterion than simple amino acid homology, the following experiment was done. The phage SP6 transcription system was used to transcribe mRNA from the four individual Torpedo subunits and from the mouse δ. When the four Torpedo subunit specific mRNAs were injected into Xenopus oocytes, functional receptors appeared in the oocyte membrane. If the β or γ subunit RNA was omitted, no response to acetylcholine was detected, while a small response was detected if the δ subunit RNA was omitted. When mouse δ specific RNA was injected in place of the Torpedo δ, a 3-4 fold larger response was measured in response to acetylcholine under voltage clamp conditions. The replacement of Torpedo γ RNA with mouse δ RNA gave no detectable response. Surface binding of α-bungarotoxin was not significantly altered by exchanging the δ subunits, which indicates that the difference is intrinsic to the channel rather than a matter of stability or synthesis rates. Examination of the amino acid sequences of the two δ subunits and the Torpedo γ did not identify an obvious region of subunit specific homology. The amino acid features necessary to determine a specific subunit are not obvious from simple homology comparisons.

We have constructed a series of chimeric subunits to try to localize subunit determining regions of the acetylcholine receptor polypeptides. Each chimera was tested in the oocyte system by replacing its RNA for each of the parent RNAs in turn. None of the chimeras we have constructed retained enough of either parental subunit characteristics to function fully in place of that parent subunit to form an acetylcholine receptor that is responsive to acetylcholine. We conclude that a minimum of two subunit-specific regions are widely dispersed over the subunit length. These data are also consistent with the conclusion that there are no discrete regions that determine subunit identity, but instead that this information is rather evenly distributed along subunit length. In some combinations, the chimeras were incorporated into surface AchRs, although these complexes were only weakly responsive to Ach. We further conclude that there are regions needed for efficient function of these subunits that are not necessary for the formation of surface complexes. We have demonstrated that the α subunits of both mouse and chick form functional receptors in the Xenopus oocyte system in combination with the β and γ subunits from Torpedo and a δ from either Torpedo or mouse. The responses of these hybrid AchRs are smaller than the response from the Torpedo AchR. In contrast, the mouse γ subunit did not form functional AchRs in any combination of the subunits mentioned above.

The present author spent the early part of her career studying the molecular biology of the actin genes of Drosophila melanogaster. Portions of each of the six actin genes were sequenced. These sequences revealed that the amino acid sequence of actin is highly conserved but that the positions of introns in these genes are strikingly nonconserved. Further, each of the Drosophila actins resembles the cytoplasmic isoforms from vertebrates, while none resemble the muscle isoforms.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Molecular Biology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Molecular Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Davidson, Eric H.
Thesis Committee:
  • Davidson, Eric H. (chair)
  • Lester, Henry A.
  • Meyerowitz, Elliot M.
  • Wold, Barbara J.
  • Benzer, Seymour
Defense Date:15 May 1986
Funders:
Funding AgencyGrant Number
NIHUNSPECIFIED
Jean Weigle Memorial FundUNSPECIFIED
Record Number:CaltechTHESIS:04152019-153632089
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:04152019-153632089
DOI:10.7907/nvv4-qt08
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/sqb.1983.048.01.010DOIArticle adapted for Chapter 2.
https://doi.org/10.1073/pnas.81.24.7970DOIArticle adapted for Chapter 3.
https://doi.org/10.1073/pnas.82.14.4852DOIArticle adapted for Chapter 4.
https://doi.org/10.1016/0092-8674(81)90506-7DOIArticle adapted for Appendix.
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11465
Collection:CaltechTHESIS
Deposited By: Melissa Ray
Deposited On:15 Apr 2019 23:06
Last Modified:16 Apr 2021 22:32

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