PTW - Cholinergic System Flashcards
(23 cards)
Q: What is acetylcholine, and what types of receptors does it act on? (2)
- A neurotransmitter involved in signalling in both central and peripheral nervous systems
- Acts on two receptor types: nicotinic (ionotropic) and muscarinic (metabotropic) receptors
Q: What are key features of nicotinic receptors? (4)
- Ionotropic receptors (ligand-gated ion channels)
- Found in both sympathetic and parasympathetic nervous systems
- Produce fast responses (microseconds to milliseconds)
- Activated by nicotine; inhibited by α-bungarotoxin
Q: What are key features of muscarinic receptors? (4)
- Metabotropic receptors (G-protein-coupled)
- Found mainly in the parasympathetic system
- Produce slow responses (milliseconds to seconds)
- Stimulated by muscarine; inhibited by atropine
Q: What are some key components needed to isolate and study receptors? (2)
- Use of a selective, high-affinity ligand to bind the receptor specifically
- A rich source of receptor protein for effective isolation
Q: What are two main techniques to study receptors? (2)
- Labelled ligand + tissue to identify and locate receptors
- Imaging methods to visualise receptor activity
Q: What are important steps in receptor characterisation? (3)
- Use labelled receptors to determine binding properties
- Perform solubilisation/purification for isolation
- Conduct biochemical/biophysical/structural analyses to study function
Q: What genetic approaches can help study receptors? (3)
- Determine a partial sequence as the first identification step
- Screen cDNA libraries for homologous sequences
- Sequence positive clones to predict the primary structure
Q: What functional studies are used to investigate receptors? (2)
- Reconstitution assays to study receptor activity after purification
- Expression studies to confirm receptor production in biological systems
Q: What are two applications of receptor study techniques? (2)
- Stable transfection of cells for drug screening
- Use of bioinformatics to identify receptor profiles and structures
Q: What is Edman degradation, and what is it used for? (2)
- A sequencing method that removes one amino acid at a time from the N-terminus
- Best for peptides up to \~30 amino acids, and improved by cleaving proteins into fragments
Q: What is mass spectrometry, and how is it used in sequencing? (3)
- A top-down workflow where the entire protein is fragmented inside the spectrometer
- Measures mass-to-charge ratio (m/z) of peptide fragments
- Results shown as a mass spectrum, revealing sequence from peak patterns
Q: How do you go from a partial protein to a full cDNA sequence? (3)
- Use Edman degradation to obtain a short amino acid sequence
- Match to mRNA codons, then screen cDNA libraries for matches
- Sequence positive clones to predict the full protein’s primary structure
Q: What are the main steps in cloning receptors using cDNA libraries? (6)
- Extract mRNA from target tissue
- Use reverse transcription to produce cDNA with polyT primer
- Insert cDNA into plasmids
- Transform bacteria with plasmids
- Screen bacterial colonies with labelled probes
- Identify positive clones for sequencing and analysis
Q: What insights can be gained from protein sequence data? (3)
- Homology with other receptors or subunits (functional and evolutionary info)
- Hydropathy plots (e.g. Kyte-Doolittle) predict hydrophobic transmembrane domains (M1–M4)
- Identify potential post-translational modifications (PTMs) that affect receptor function
Q: What are structural features of nAChR subunits? (3)
- 4 transmembrane domains (M1–M4), mostly α-helices
- Extracellular domain with β-sheet structure
- Disulphide bonds (e.g. Cys192/193) stabilise structure
Q: What are four common membrane protein expression systems? (4)
- Xenopus oocytes – support high expression and electrophysiology
- E. coli – efficient but lacks post-translational processing
- Insect cell culture – allows proper folding and PTMs
- Mammalian tissue culture – ideal for expressing human-like membrane proteins
Q: What are challenges of expressing eukaryotic proteins in E. coli? (6)
- Different codon usage
- No endoplasmic reticulum or PTMs
- Different insertion machinery for membrane proteins
- Altered lipid composition
- Risk of inclusion body formation
- Difficult to assemble multi-subunit proteins
Q: What are examples of traditional and modern detergents for solubilising membrane proteins? (3)
- Traditional: ionic (e.g. SDS), zwitterionic (e.g. CHAPS), non-ionic (e.g. DDM)
- Modern: amphipols and nanodiscs (e.g. SMALPs)
- Choice depends on desired outcome: structure, activity, or reconstitution
Q: What are two common methods of purification for membrane proteins? (2)
- Tag-based affinity chromatography (e.g. His10/12, MBP)
- Ligand affinity chromatography (e.g. α-bungarotoxin for AChR)
Q: How can tag-based and ligand affinity chromatography be combined? (2)
- Use tag-based purification for crude isolation
- Follow with agonist-affinity column for functional receptor selection
Q: How are receptors reconstituted into lipid vesicles? (3)
- Detergents exist in equilibrium between micelle and monomer
- Remove detergent slowly (via dialysis, Biobeads, or dilution)
- This forms proteoliposomes — artificial vesicles with inserted protein
Q: What are key features of the acetylcholine binding site on nAChR? (3)
- Rich in aromatic residues (not negatively charged)
- Located near the vicinal disulphide bond Cys192/Cys193
- Binding occurs at the interface between subunits (α/δ and α/γ)
Q: What method is used to determine the structure of nAChR? (1)
- Cryo-electron microscopy (Cryo-EM) of helical arrays
