Receptors and fusion proteins Flashcards
What are some features of ionotropic receptors?
Ligand gated
<50ms speed
All or nothing
Multiple interchangable subunits
Neurotransmitter sensor
Changes membrane potential
What are some features of metatropic receptors?
GPCR
100ms to minutes
can amplify/dampen signals
Generally monomeric
Describe glycine receptors
Fast inhibitory neourotransmission at synapses
Motor control and pair processing in CNS
5 subunits
Inhibited with picrotoxin and imaged without ligands
Glycan density at Asp62 affects how the protein affects ER
Describe nAchR structure
Cys-loop ion channel
Pentameric
Lots of TM helices, hydrophobic outside, hydrophilic inside
Ach binds two sites at interfaces of each α subunit
C-loop closes around the ligand
How are Cl- channels gated?
Glu receptors which move Cl- out of the cell to inhibit polarisation of the membrane
What helps GluCl crystallise?
Fab antibody fragements bind the channel at 5 sites which are (+)
How does the GluCl channel open?
β1-β2 loops twist and tilt M2 and M3 away from the central axis, opening the core
What is transducin?
GPCR which has rhodopsin as its G-protein
What is rhodopsin made up of?
Opsin and retinal (chromaphore)
Describe transducin activation
- One photon activates retinal which gets transducin in an activated state
- Gα-GDP is exchanged for Gα-GTP
- GTP is hydrolysed to switch off the signal
- Membrane is hyperpolarised by Na+ import and Ca2+ export which is interpreted by the brain as light
What structural features do transducin have?
Amphiphilic domain near C-terminus
Conserved sequences on TM3 and TM6/7 form an ionic lock.
TM6 swings open when light is absorbed, allowing Gαt
At ground state a lid prevents access to a rhodopsin binding site, gated by ionic lock between TM6 and 7
What happens when retinal absorbs light?
Energy pushes against the protein, breaking the ionic lock and allowing rhodopsin to bind to transducin
11-cis to all-trans retinal
Gt preferrentially binds Meta II to induce transducin activity
How are transducin intermediates trapped for study?
- Retinal removed, add Gαt fragment to have Meta II conformation
- Mutants with consituitive activity, e.g disrupting the counterion to keep it in an all-trans conformation
How are α2A adrenergic receptors targetted for CNS pain relieving effects.
Helical movements trap Gα in the active state
Using a virtual molecule library (ZINC15) which scores molecules based on van der waals and electrostatic interactions
How are β2AR locked in an open conformation?
Carazolol is an inhibitorwhich sits in a binding site with low Kd and slow off rate
Describe β2AR activation
- Adrenaline binds to the β2AR. TM5 and 6 open up which allows G protein to bind
- Gs activates adenylate cyclase, converting ATP to cAMP
- cAMP activates L-type Ca2+ channels on the ER to release Ca2+
- PKA is activated, eliciting downstream effects
What inactivates β2AR?
BARK (type of GRK)
Arrestin
PKC
Describe the nuclear pore
Found at inner and outer junctions of nuclear envelope
30 different nucleoporins
Aids in DNA histone synthesis in cytosol
Expands when a peptide sequence is recognised
Transport guided by exportins, importins and Ran-GTP
What are fusion proteins used in?
Fusion of organelles
Plasma membrane repair
Vacuole fusion
Viral entry
Exocytosis
What forces prevent membranes spontaneously fusing?
Membrane proteins repelling each other
Polar lipid heads repelling each other and attracting water (repulsive hydration force)
Membrane movement creating an entropic barrier
Outline how two haemaglutanin molecules form a membrane pore in the host cell membrane
- Haemaglutanin overcomes energetic barriers and recognises cell receptors. The receptor is taken up by the endosome via endocytosis
- pH5 in the endosome causes haemaglutanin heads (HA1) to become loosely tethered. α helix forms and kicks the N-terminus to the host cell membrane
- HA0 is proteolytically cleaved to HA1 and HA2 for membrane fusion.
- Pore forms
Why is haemaglutanin proteolytic cleavage irreversible?
Free energy is low after cleavage
How is the SARS COV-2 spike protein embedded into host cell?
TMPRSS enzyme from host cell cuts spike protein which reveals hydrophobic amino acids that allow it to embed into the host cell membrane
