Channels & Receptors Flashcards
Name 3 Ion Channel functions
- Transport Ions across a membrane (Secretion/absorption of fluids)
- Regulation of membrane potentials (nerve/muscle cells)
- Ca2+ influx into cytoplasm (secretion/muscle contraction)
Common structural features of ion channels
TM proteins of 2 or more alpha-helices, 2-6 subunits surround pore (except for cl, H2O and NH3 channels). Classified into subgroups based on gating mechanism + ion selectivity which is defined by size and amino acids lining pore
Tell me about KscA
Simple K+ Ion channel.
4 Identical subunits, 2TMH + P-Loop
TMHs tightly packed on cytoplasmic side
Gate controlled by membrane potential, mechanic stress & ligands
Tell me about voltage gated ion channels
Similar structure to Ion channels but with an S1-4 voltage sensing domain, large polypeptides extend into cytoplasm, has a plugging mechanism. Na+/K+ channels create APs in excitable cells.
Tell me about KvaP
S1-4 form a voltage sensing domain, S3-4 paddle contains Arginine residues that sense changes in membrane potential. S4-5 linker connects the pore domain to the voltage sensing domain. Standard S5-PLoop-S6.
Tell me about ligand-gated channels
Similar structure to voltage-gated channels but requires ligand-binding (extra or intracellularly). Two common domains are:
1. Calmodulin, bound via the N-terminus it binds Ca2+
2. Cyclic Nucleotide-binding domain, bound via the C-terminus.
What is nAChR?
It is a cys-loop type receptor, it is pentameric, each subunit containing 4TMHs (M1-4) with an extracellular domain recognising and binding ligands or NTs such as GABA A, nicotinic acetylcholine and serotonin.
In muscles, it is composed of 5 subunits of either α, β, γ and ε.
Tell me about neuronal nAChR
Neuronal nAChR contains α2-10 and β2-4 subunits, each with a different affinity depending on composition and location. α4β2 is abundant in the cortex and hypothalamus.
Chronic exposure to nicotine and varenicline agonists leads to receptor upregulation.
Polymorphisms in α4 and α6 subunits have been linked to tobacco dependence but improved cessation.
Rare variants can even confer protection against tobacco dependence.
What is ADNFLE?
Autosomal Dominant Nocturnal Frontal Lobe Epilepsy. 9 mutations in the M2 region of the neuronal α4 subunit have been identified.
Leads to enhanced receptor function and increased nicotinic-mediated transmitter release causing ADNFLE seizures.
Describe glutamate receptors, give examples
They bind the ligand glutamate. They are tetrameric; dimer of dimers with a ligand-binding site within a cleft which closes upon ligand-binding. They are vital to brain function, dysfunction contributes to many diseases.
a) AMPA receptor: mediates fast excitatory synaptic transmission in the CNS
b) NMDA receptor: involved in learning and memory, slower than other isoforms
c) Kainate receptors: similar to AMPA but with a lesser role at synapses, linked to schizophrenia, depression and huntingtons disease
What is the role of RNA processing in AMPA subunits?
AMPA subunits exist as one of two forms; flip or flop. Alternate splicing of 2 exons in the primary transcript leads to 2 different proteins with different domains in the extracellular loop. Flop has a faster desensitization rate and reduced current responses to glutamate than Flip.
Tell me about RNA editing enzymes role in AMPA receptors
GluA2 Q/R site located on the M2 subunit inside the channel pore is key to the functionality of AMPA receptors. An RNA Editing enzyme converts a CAG to a CCG; a glutamine residue to an Arginine making the channel impermeable to calcium. Mutant mice lacking this enzyme were prone to seizures and early death.
What are P2X receptors?
They are ATP-gated channels, they require 3 ATP to open. Trimeric proteins, each subunit has 2 TMHs and a large extracellular domain.
There are 7 types of P2X receptor, their dysregulation can lead to disease;
a)P2X2 dysregulation leads to hearing loss
b) P2X4 dysregulation can lead to pain
c) P2X7 dysregulation can lead to inflammation and neurodegenerative disease
How is platelet aggregation stimulated?
Thrombin cleaves part of the N-terminus of protease activated receptors (PAR) on platelets, the new N-terminus sequence acts as a tethered ligand activating PAR. This signalling pathway leads to the crosslinking of platelets. Different receptors cooperate to elicit the aggregation of platelets. Collectively, they can respond to thrombin, exposed basal lamina and ADP.
What does Gαi do?
It responds to α-adrenergic amines, acetylcholine, chemokines, tastants and neurotransmitters.
It inhibits adenylyl cyclase, opens K+ channels and closes ca2+ channels.
It is involved in negative feedback in synpases, and decreases pancreatic insulin.
What does Gαq do?
It responds to α-adrenergic amines, acetylcholine and neurotransmitters.
It activates phospholipase Cβ to produce IP3 and release ER Ca2+.
It is involved in smooth muscle contraction and vasoconstriction.
What does Gαs do?
It responds to β-adrenergic amines and hormones.
It activates adenylyl cyclase to produce cAMP.
β1 a.a. leads to increase heart rate. β2 a.a. leads to smooth muscle relaxation.
What does Gαt do?
It responds to rhodopsin.
It activates cGMP phosphodiesterase; cGMP hydrolysis.
It is involved in phototransduction in photoreceptor cells.
What does Gα13 do?
It responds to Thrombin.
It activates a Rho GTPase.
It is involved in platelet activation.
What does Gαolf do?
It responds to odorants.
It activates adenylyl cyclase to produce cAMP.
It is involved in our sense of smell.
Specificity of odorant detection lies in GPCRs with specific ligand-binding sites.
What are GPCRs?
GPCRs are the most diverse type of receptor able to respond to physical and chemical stimuli. They have 7 transmembrane helices with an N-terminal extracellular ligand-binding domain and a C-terminal heterotrimeric g-protein binding domain.
Describe direct activation of ion channels
Activated GPCRs act as GEFs, swapping Gα GDP for GTP, GαGTP binds to ion channels regulating them. This is often slower, and channels can remain open or closed for longer, minutes rather than millisecods.
List some 2nd messengers Gproteins regulate
Hydrophobic lipids which remain confined to the membrane.
Small soluble molecules which can diffuse through the cytoplasm such as cAMP and cGMP.
Ca2+ ions
All are key in signal amplification.
Tell me about how Vibrio cholera and Bordetella pertussis interact with G proteins
Vibrio cholera toxins inhibit GTPase activity of the Gαs subunit leaving Adenylyl cyclase constitutively active, producing cAMP. Cells release Cl-, Na+ and H2O leading to diarrhoea and extreme dehydration.
Bordetella pertussis toxins inactivate inhibitory Gαi reducing the inhibition of Adenylyl cyclase; too much intracellular cAMP erodes respiratory epithelium via the excess release of mucus. This also triggers coughing fits.
Tell me about Uveal Melanomas.
They are cancerous growths in the eye arising from neural crest cells. Over 90% of cases are associated with mutations in the Gαq subunit, as a result, GTP hydrolysis does not occur leaving the melanin growth pathway constitutively active.
Describe how cAMP is stimulated as a 2nd messenger
Adenylyl cyclase is inhibited by Gαi and activated by Gαs
1. Ligand binds to a receptor, activating it’s G-protein
2. G-protein moves along membrane, binds to and activates adenylyl cyclase
3. Adenylyl cyclase catalyses the conversion of ATP to cAMP
4. cAMP phosphorylates/activates Protein Kinase A (PKA)
5. PKA phosphorylates/activates proteins
6. Proteins stimulate a cellular response
Describe the role of β2-adrenoceptors in metabolic regulation
In the liver and skeletal muscle, β2-adrenoceptors are key in glycogen lysis:
1. Ligand binds to β2-adrenoceptor activating Gαq
2. Gαq activates adenylyl cyclase, stimulating cAMP production
3. cAMP activates PKA
4. PKA triggers a signalling cascade
5. Glycogen phosphorylase is phosphorylated and is now active
Tell me about cGMP
cyclic GMP is produced by guanylyl cyclase which is either receptor-bound or free in the cytoplasm. Local [cGMP] is determined by rates of production, diffusion, and removal. Messages are encoded in [cGMP] and the frequency of changes in [cGMP]. It is removed by phosphodiesterases
Tell me about the role of lipids as 2nd messengers
Membrane lipids can be targeted by receptor-regulated lipases to generate two types of 2nd messenger, such as PIP2 by Phospholipase Cβ to generate:
a) IP3, a water soluble molecule which diffuses through the cytoplasm and binds to IP3 receptors (ca2+ channels on ER inner membrane)
b) DAG, hydrophobic molecule which remains in the membrane and activates Protein Kinase C
Lipid Kinases can add phosphate groups back to lipids, e.g. DAG to phosphatidic acid, PI to PIP, PIP2, PIP3
Where does specificity arise from in PKC; serine/threonine kinases
They vary in their regulatory domains, the C2 domain allows lipid-binding, C1 domain allows DAG binding..
Other key domains are:
X/Y catalytic domains which remain largely unchanged
PH domains which bind λ/β G-protein subunits
Large Carboxyl domain binds G-protein α-subunit
In research, what is used as an analogue of DAG to activate PKCs
PMA phorbol ester
How does calcium concentration vary in a cell.
In a resting cell;
a) Cytosolic [Ca2+] is ~100nm, maintained by ATP-driven Ca2+ pumps
b) Extracellular [Ca2+] is ~2.5mM
c) ER Lumen [Ca2+] is ~ 400µm known as the Calcium Store
How is the calcium store refilled?
After muscle contraction, or synaptic transmission, calcium levels in the ER lumen can decrease, this stimulates a conformational change in ER lumen receptor STIM, exposing a cytoplasmic domain which interacts with ORAI, a cell membrane calcium channel, opening it allowing the refilling of the calcium store.
Loss of function mutations in ORAI result in severe combined immunodeficiency.
Tell me about the desensitization of GPRs
a) GRKs; GPCR receptor kinases prevent g-protein binding
b) β-Arrestin internalises the GPCR for degradation or recycling.
Overstimulation of GPCRs can lead to cancer or tachyphylaxis (decreasing responses to drugs)
