Ion channels structure and function Flashcards
What are ion channels
Integral membrane proteins that have ion-permeable pores that regulate current flow across the membrane. They change cell excitability (Na+ and K+) and Ca2+ activates internal signaling pathways.
Electrochemical gradient definition
the combination of charge difference and ion concentration difference across a membrane. Nernst equation calculates the membrane potential that would cause flow reversal.
What are the 3 key components of ion channels and their role
Sensor - picks up stimuli, e.g., ligands/proteins binding, voltage change, light, temp, mechanical force, H+ ions
Gating mechanism - changes structure of ion channel to open the pore.
selectivity filter - the AA structure in the pore region of the channel, determining which ions can permeate.
What are additional factors that can affect ion channel function
Desensitisation/inactivation - closed or subconductance state following activation.
Subunit composition - homo vs heteromeric. different combinations lead to different effects.
PTM - phosphorylation regulates trafficking, localistation and internalisation.
Auxiliary subunits - modulatory proteins that can bind and regulate trafficking (how they reach the membrane), density (how they are clustered), localisation (to particular regions), and kinetics (alters desensitisation).
What is electrophysiology
an experimental technique that allows for the real time and accurate measurement of current flow and potential changes across a membrane. Uses an electrode.
Mechanism for pore opening in VGICs and LGICs
VGICs: the TM (+ve) paddles are on the (-ve) cytosolic face when the pore is closed. They flip to the extracellular face when the cell interior is depolarised (more +ve), with the electrical repulsion leading to a conformational change in the pore, enabling the movement of ions through it.
LGICs: upon agonist binding the TM domains undergo a conformational change allowing the ion to pass through the pore. ACh binds to nAChRs extracellularly, cyclic nucleotides bind to the cytosolic region of CNG channels.
classes of iGluRs and how they were named
AMPA, Kainate, NMDA
Named after the selective agonists that were used to identify them.
Structure of iGluRs
Tetramer
S1/2 - ligand binding domains
structure:
Amino-terminal domain (ATD) - S1 - M1 - M2- M3 - S2- M4 - Carboxyl-termincal domain (CTD)
All M domains are TM
Subunit combination determines selectivity, pharmacology, kinetics, and modulatory sites.
AA sites in selectivity filter for iGluRs
Q/R/N site found.
AMPA and kainite have Q/R AA sites, while NMDA contains N sites, which enables NMDA to be much more permeable to CA2+.
Mutations can lead to disrupted ion channel selectivity/function
Examples of structural analysis of ion channels before 3D structuring
Primary structure mapping is gene sequencing identifying all the AAs in the ion channel which can then be used to plot several characteristics of the channel, e.g., Hydrophobicity plots which identify the regions most likely to be TM domains.
Site-directed mutagenesis selectively changes individual residues to see how it impacts channel function.
History of 3D channel imaging
Crystallography was done on purified samples of lipid-bound bacterial potassium channels.
Cryo-EM: snap frozen protein samples are examined under electron microscopy (EM), with multiple proteins averaged to get a high-resolution image of the channel.
Later EM mapping was done on pore opening of an nAChR.
Molecular dynamics (MD) simulation and Markov state modelling (MSM) combined with site-directed mutagenesis was used to deduce mechanisms of voltage sensing and gating in VGCCs.
Optogenetics in mice
Light-activated ion channels from archaea, bacteria, fungi, or algae contain a chromophore, which activates the channel depending on the wavelength of light absorbed.
The opsins (photoreceptors) have been engineered to have different wavelength sensitivity, kinetics, and expression.
2 opsins were transfected in vivo into mice neurons as colocalised (put in together) bidirectional (one stimulates ion movement, other inhibits) optogenetic modulators. This enabled the experimenters to stimulate or inhibit the neurons of mice using a probe that contained red and blue lights.
one colour stimulated neurons, while the other inhibited.
