Ligand-gated ion channels Flashcards
what does selective expression of receptors and ligands allow?
In multicellular organisms, selective expression of certain receptors and the molecules involved in signal transduction allow cells to respond specifically to particular stimuli
what are ion channels?
- A channel is a transmembrane protein that transports molecules from one side of the membrane to the other
- They are specific e.g. Na+, K+ or Cl-
- They can be open (non-gated) or use carries (gated)
what are the 3 essential functions of ion channels?
- transport ions across the membrane: secretion/absorption of fluids
- regulate membrane potentials e.g. in nerve and muscle cells
- calcium influx into cytoplasm: secretion and muscle contraction
what are the main structural features of all ion channels?
- transmembrane proteins made up of 2 or more alpha-helices crossing lipid bilayer (transmembrane domains)
- 2-6 subunits surround the pore
what are the exceptions to the main features of ion channels?
exceptions are Cl-, water and ammonia ion channels which each have a pore existing in the middle of a single subunit
how are ion channels classified?
- their gating mechanism: voltage or ligand
- the ion selectivity of the pore - defined by physical size of the pore and the amino acids that line the pore
what is an alpha-helix?
- secondary protein structure
- has a right-hand helix conformation
what is a beta-sheet?
- secondary structure
- beta strands are connected laterally by at least 2 or 3 backbone hydrogen bonds
- forms a sheet
what are subunits?
- single proteins which join with others to make a protein complex
what is a transmembrane domain (TM)?
- protein that spans the width of the membrane from extracellular to intracellular sides
- usually helical in shape
what is a p-loop/pore?
the pocket in which the ion will bind
how have ion channels evolved?
- 400 genes in humans code for membrane channels
knowledge of structure reveals evolutionary relationships:
- The pH-regulated K+ channel KcsA from Streptomyces lividans serves as a model for all channels
what is the structure of a simple ion channel e.g. K+ channel?
- TM helicase structures form a p-loop pore which is highly selective
- 4 subunits, 2 TMs
- on cytoplasmic side, TMs are tightly packed to form a gate
- controlled by membrane potential, mechanical stress and ligands
what are the two main functions of voltage-gated ion channels?
- Na+ and K+ create APs in excitable cells
- Ca2+ is transported into cytoplasm where second messengers elicit cellular responses
what is the structure of voltage-gated ion channels?
based on similar structure to simple ion channel except:
- additional helices S1 and S4 form a voltage-sensing domain lateral to the subunits
- large polypeptides extend into cytoplasm
- plugging mechanism by voltage-sensing domains - allows channel to be gated by voltage
- 4 subunits
- 6-24 TMs
- contains p-loop, cytoplasmic anchors and plugging mechanism
example: voltage-gated potassium channel
what are Transient Receptor Potential (TRP) channels?
- share common structural features with voltage-gated ion channels
- evolved to sense chemicals and physical stimuli
- 4 subunits
- 6 TMs
- responds to hot/spicy taste
- contains P-loop, cytoplasmic anchors and plugging mechanism
- no voltage-sensing domain
what is the structure of ligand-gated ion channels?
similar structure to voltage-gated but controlled by ligand-binding:
- Transmembrane domains, binding domain faces out of the cell, proteins come together to form heteromeric/homomeric receptor
- Transmembrane domains form aqueous pore for ions to flow through
- When ligand binds, aqueous pore opens so that ions can move across gradient
o- Flow of ions generate a current which leads to changes in membrane potential
- 4 subunits, 6 TMs
- Channel gating is controlled by ligand binding
- has no plugging mechanism and no voltage-sensing domain
give an example of a ligand-gated ion channel and describe its structure:
cyclic nucleotide-gated ion channel: senses cAMP or cGMP
- tetramer: 4 subunits
- 6 TMs
- S5 and S6 alpha-helical domains line central pore with p-loop controlling filter
- addition of regulatory domains to intracellular N and C terminals
- cyclic nucleotide-binding domain on C terminal opens a pore permeable to Na+ and Ca2+
- ligand must bind to 3 of the 4 sites for the channel to open, lose 1 and channel closes to give sharp conc-response curve
- calmodulin is bound to N-terminal. if calcium binds to N-temrinal, calmodulin provides negative feedback
what are extracellular ligand-gated ion channels known as?
form distinct family of ionotropic receptors:
- Cys-loop type
- ionotropic glutamate type
- P2X type
- calcium-release type
what is the general function of ionotropic receptors?
- TMs regulate specificity of channels: if positively charged, they let anions through (Cl-), if negatively charged they let cations through (Na+, K+, Ca2+)
-Na+/K+ selective channels control membrane excitability - channels with added permeability to calcium indirectly regulate activity of calcium-sensitive proteins
- chloride-selective channels control membrane excitability by hyperpolarisation to reduce AP firing
what is the structure of the Cys-loop type ionotropic receptor? give an example of this receptor type:
- pentameric assembly: 5 subunits, 4 TMs
example: nicotinic acetylcholine receptor (nAChR)
- 5 subunits: alpha, beta, gamma and epsilon (1 can repeat)
- subunit composition can change -> selectivity of ions that can flow through
- each subunit has 4 TMs (M1, M2, M3, M4)with a large externally-facing N domain
- intracellular loop between M3 and M4
- M2 lines the pore and determines ion specificity
what do the variety of subunits within ionotropic receptors achieve?
- Different subunit combinations make up receptors in different parts of the brain
- Complexity provides diversity and opportunity for drug targeting
E.g. nAChR-alpha4 subunit is involved in reward pathways and nicotine addiction
how can targeting nAChRs help to treat nicotine addiction?
neuronal nAChRs exist as alpha2-10 and beta2-4, and each has different affinity depending on subunit composition and location:
- alpha4-beta2 are expressed in cortex and hippocampus and have high affinity to agonists nicotine and varenicline
- chronic exposure to nicotine leads to nAChR upregulation
- we can target these alpha4-beta2 receptors specifically, without affecting the other nAChRs that have different subunit compositions
- genetic studies show polymorphisms in CHRNA4 (alpha-4) and CHRNA6 (alpha-6) are linked to tobacco dependence but better cessation outcomes - can personalise treatment options
- rare variants are protective against nicotine dependency
what mutation causes ADNFLE (autosomal dominant nocturnal frontal lobe epilespy)?
mutation in the M2 region of the human alpha-4 subunit (CHRNA4) of nAChR causes ADNFLE
- 9 mutations have been identified
- receptors have a delayed response to ACh - lag caused by the slow unblocking and recycling of receptors
- the ACh concentration dependence of use-dependent potentiation and delay in rising face is caused by slow unblocking of closed receptors
- this means there is an enhanced nAChR activity, so increased nicotinic-mediated transmitter release, leading to ADNFLE seizures
what is the structure of ionotropic glutamate receptors?
- tetrameric assembly: 4 subunits, 3 TMs
- pore is inverted
- activated by glutamate
- forms as a dimer of dimers: the ligand-binding site is in a cleft that closes when occupied
why are ionotropic glutamate receptors important?
vital to every aspect of brain function
- dysfunction of these receptors contributes to human disease
how are ionotropic glutamate receptors diverse? what subcategories do they have?
Multiple genes, alternative splicing and RNA editing contribute to diversity (pharmacology, permeability and function) of glutamate receptors:
- AMPA = mediate fast excitatory synaptic transmission in CNS
- NMDA (N-methyl-aspartate receptor) = involved in learning and memory (slower than other isoforms)
- Kainate = similar to AMPA but lesser role at synapses -> linked to schizophrenia, depression and Huntington’s
how does RNA splicing have consequences in AMPA receptor subunits?
- Each subunit exists as two splicing isoforms - flip & flop
- Alternative splicing of two exons in the primary transcript = two protein isoforms with different domain in the extracellular loop
- Causes them to have different kinetic properties: Flop = faster desensitization rate and reduced current responses to glutamate than flip
how does RNA editing have consequences in AMPA receptor subunits?
- The GluA2 Q/R site is located in the M2 of the subunit, inside the channel pore.
- CAG (glutamine) codon to a CGG (arginine) codon
- Effect of GluA2 Q/R editing on channel Ca2+ permeability – stops Ca2+ entering the cell
- Mutant mice lacking enzyme responsible for RNA editing prone to seizures and early death
what is an example of dysfunction in NMDA receptors?
- The NMDA receptor is thought to be important for controlling synaptic plasticity and mediating learning and memory functions
- Excess stimulation of NMDA in stroke leads to neuron death
how can dysfunction of RNA modification lead to pathogenic conditions in ionotropic glutamate receptors?
Downregulation of GluA2 Q/R editing in the motor neurons of ALS patients = increase in Ca2+ permeable AMPA receptors causes damage due to glutamate excitotoxicity
- Downregulation of the editing enzyme ADAR2 not a mutation in the editing site
In glioblastoma, decreased ADAR2 activity correlated with increased malignancy
- Increase in Ca2+ = Akt pathway promoting proliferation and tumorigenesis
- This was reversed when GluA2 Q/R was edited
Potential targets for therapeutic applications
what is the structure of P2X ionotropic receptors?
- trimeric assembly: 3 subunits, 2 TMs
- ATP-gated ion channel
- large extracellular domain
- 3 ATP molecules are needed to open the channel
- Widely expressed
- P2X1-7 subtypes of subunits
why are ionotropic receptors good drug targets?
they have diverse and specific subunits so drugs can be made to specifically target those subunits, meaning other ionotropic receptors without those subunits will be unaffected
give an example of how dysfunction in receptor expression leads to physiological conditions:
in macrophages, activation of caspase 1 inflammasome results in release of cytokines for inflammation
- These receptors for caspase 1 can be targeted to treat inflammation
