PHSI3009: Module 3 - Protein Structure & Function

Integral membrane proteins are responsible for…

Transport across the membrane

What is cloning and site-directed mutagenesis?

Using bacteria in the lab as factories to produce DNA for research use

• done using a plasmid or vector

• Use PCR to change AAs to desired protein of interest

• Site-directed mutagenesis: introduce a single point mutation and investigate functional effect

  • start by designing primers that introduce mutation

  • confirm mutation using sequencing

  • express protein and check function of altered AA using chosen experiment

What are protein sequence alignments?

Aligning AAs between closely related proteins to provide information about the structural basis for differences between closely related proteins.

This information can be used to predict which AA residues are responsible for functional differences between the proteins:

• substrate specificity

• inhibitor specificity

• test predictions from structural models

What is electrophysiology?

Using ion flux to change the electrical potential of a cell and generate a current

• measure electrical currents associated with function of protein

• activation of GPCRs can trigger stimulation/inhibition of ion channels

What is the challenge of working with membrane proteins?

They are difficult to remove from the membrane (natural environment), so we need detergent to solubilise membrane proteins

• note: most membrane protein structures are of prokaryotic origin

How is protein purified?

For structural biology, protein needs to be pure (homogenous), stable, soluble, and of high quantities

Example procedure: Gel filtration (size exclusion chromatography

Then, functional assays can be conducted

What are some methods used in structural biology?

Structural biology is the study of the molecular structure and dynamics of biological macromolecules, particularly proteins and nucleic acids. Some methods used to study this are:

1. X-ray crystallography: Provides detailed information on protein interactions with ligands, cofactors, and ions, but requires 3D crystals

2. Cryo-Electron Microscopy (cryo-EM): Analyses macromolecule structures in their native environment, advancing rapidly, especially for membrane proteins, but limited by molecule size.

3. Solution and Solid-state NMR: rapidly advancing, but sensitivity and resolution enhancements are still needed to make this a robust technology

CryoEM and (A - technique) can be used to visualise (B).

This helps us (C - 2 things)

A - protein crystallography

B - protein structures at very high resolution (atomic scale)

C - understand conformational changes in proteins AND develop drugs to specifically bind to proteins

How does protein crystallography work?

Vapour diffusion (controlled evaporation)

• crystal contains many molecules in an ordered 3D array

• protein crystallography uses X-rays

What is a synchrotron?

A synchrotron produces light by accelerating electrons almost to the speed of light

• infrared, UV, and X-rays are sent down pipes called beamlines to work areas, where scientists run their experiments

• when this bright light is aimed at a very small sample, an image of the sample’s properties is created on a detector

  • this image is sent to a computer to analyse the sample’s molecular structure

What is Cryo Electron Microscopy?

Cryo EM uses a beam of electrons to examine the structures of molecules at the atomic scale

• as the beam passes through a sample, it interacts with molecules and projects an image of sample onto detector

• Cryo EM does not require crystals, instead using frozen samples of protein (enables visualisation of molecule movement)

• Cryo EM is limited by molecule size (difficult to solve structures of molecules smaller than 100 kDa, which is larger than the average protein size)

Identify the 5 classes of the Transport Classification System

Class 1: Channels and Pores

These are proteins that allow the relatively free flow of solutes across the membrane. They are divided into five subclasses.

Class 2: Electrochemical Potential-Driven Transporters (Carriers)

These proteins bind their solutes to form complexes before transporting them across the membrane via secondary active transport processes such as symport and antiport.

Class 3: Primary Active Transporters

These include ATPases and ATP-binding cassette (ABC) transporters. Other examples are transporters driven by redox reactions or by light (e.g., photosynthetic reaction centers).

Class 4: Group Translocators (not covered in module)

These transporters enable the phosphorylation of sugars during their transport into bacterial cells.

Class 5: Transmembrane Electron Transfer Carriers (not covered in module)

These include one-electron and two-electron carriers involved in electron transport across membranes. Redox proteins in this class are often not classified as transport proteins.

Identify ATP-dependent transporters (4)

1. P-type ATPases: found in plasma membrane and include the Na⁺/K⁺-ATPase and the Ca²⁺ pump (for regulating calcium in the muscle and heart)

2. F-type (e.g. mitochondrial and bacterial ATP synthases)

3. V-type: maintains the low pH in vacuoles in plant cells and lysosomes, endosomes, the Golgi, and secretory vesicles of animal cells

4. ABC (ATP-Binding Cassettes): nucleotide-binding domains

Features of ABC transporters (4)

• have two nucleotide-binding domains (NBD) and two membrane domains

  • membrane domains typically have 12 TM helices in either a single peptide or two subunits

• conformation of NBD of the maltose transporter (MalK) dimer varies from the resting state (with two NBDs separated from each other), ATP-bound state (closed), and the post-hydrolysis state (open)

  • each subunit has an NBD and a regulatory domain

• found in large numbers in organisms

• some are very specific, others are quite promiscuous

Note: this is just an overview, we do not need to know so much abt ABC transporters

Features of P-type ATPases (5)

• uses energy from ATP hydrolysis to drive transport

• ~75% of total ATP is used to pump in nutrients, pump out toxins, and create ion gradients

• substrates can vary from ions to phospholipids, P-type ATPases perform vital functions in prokaryotes and eukaryotes

• Their name refers to the phosphorylated enzyme intermediate formed during the reaction when the g-phosphate group of ATP is transferred to an aspartate residue at the beginning of the conserved motif DKTGT

• There are over 300 known members of this superfamily, grouped into 5 subclasses

  • Na⁺/K⁺-ATPase is in class P_2C

Features of Na⁺/K⁺-ATPase (3)

• transports 2 K⁺ in and 3 Na⁺ out for every ATP hydrolysed

• helps create typical membrane potential of -50 to -70 mV across plasma membrane of most cells

• mechanism of coupling active transport with ATP hydrolysis involves shifting form the phosphorylated form with high affinity for K⁺ and low affinity for Na⁺ to the dephosphorylated form with high affinity for Na⁺ and low affinity for K⁺

  • changes in transporter conformation and ion affinity on each side of the membrane are involved in the mechanism

Which two P-type ATPases show similar structures?

Ca²⁺ ATPase from SR (SERCA) and Na⁺/K⁺ ATPase (plasma membrane)

• both have 3 cytoplasmic domains involved in ATP hydrolysis:

  • phosphorylation (P), nucleotide-binding (N), and actuator (A) domains

• main catalytic (α) subunit α1-4

Describe the structure of Na⁺/K⁺ ATPase

• catalytic α subunit

  • TMD 1-6 transport core, TMD 7-10 support

• bitopic β subunit (brown)

  • is a 45-kDa protein with a short cytoplasmic tail, one TM helix, and a larger and highly glycosylated domain outside the membrane, where it interacts with extracellular loops of α subunit

  • trafficking to the plasma membrane

  • affects affinity for K⁺

• tissue-specific γ subunit (green; FXFD regulatory protein)

  • single TM helix, with a basic cytoplasmic C terminus and an acidic extracellular N terminus

  • finely tunes the activity of Na⁺/K⁺-ATPase, mainly by effects on the Na⁺ affinity, but also effects on K⁺ affinity and transport rates

Describe the structure of K⁺ binding sites on Na⁺/K⁺ ATPase

ATP phosphorylates an Asp residue in the highly conserved sequence DKTG only in the presence of Na⁺ (requires Mg²⁺ as a cofactor)

• aspartyl-phosphate thus produced is hydrolysed only in the presence of K⁺

• K⁺ binding sites in shark Na⁺/K⁺ as viewed from cytoplasm (see image)

• The two K⁺ ions (purple spheres) are very close, with no oxygen ligands or waters (red spheres) between them

• The AAs involved in coordinating the ions are labelled with the residue numbers for shark enzyme

What conformational changes occur during the Na⁺/K⁺-ATPase catalytic cycle (7)?

1. Na⁺/K⁺-ATPase starts with an inwardly opened conformation with access to the ion-binding sites

2. After binding of 3 sodium ions, TM1 rearranges to a position that blocks the cytoplasmic entrance pathway

3. Following sodium occlusion, D39 is phosphorylated

4. ADP is released, conformational change opens an extracellular pathway allows the exit of the 3 sodium ions

5. In the externally opened conformation, 3 ion-binding residues are directly visible from the outside

6. Binding of two extracellular potassium ions initiates closure of the extracellular gate

7. Dephosphorylation of D369

How can Na⁺/K⁺-ATPase be manipulated in medical settings?

• Na+/K+-ATPase is strongly inhibited by ouabain, a glycosidic steroid that belongs to a class of hormones called cardiotonic steroids

• Endogenous cardiotonic steroids affect renal sodium transport and blood pressure, regulation of cell growth, differentiation, apoptosis, and control of some brain functions

• Ouabain and the related compound digitoxin (brand names; Crystodigin, digoxin, and Lanoxin) have long been used to treat heart failure

• By inhibiting the Na+/K+-ATPase and thus decreasing Na+ gradients, it reduces Na+/Ca2+ exchanger activity, leaving the concentration of Ca2+ higher in the heart muscle cell to allow stronger contractions

What medical conditions can be linked to Na⁺/K⁺-ATPase (5)?

• FFXYD3 and 5 are upregulated in several cancers (functional effect not clear)

• Na+/K+-ATPase malfunctions in cardiovascular, neurological, renal, and metabolic diseases

• Hypertension; Adrenal overproduction of aldosterone is the cause of hypertension in up to 10% of hypertensive patients

• Familial Hemiplegic Migraine (FHM) is an autosomally inherited form of migraine, experiences aura and weakness in one side

  of the body during attacks

• Rapid-onset Dystonia Parkinsonism (RDP), Alternating Hemiplegia of Childhood (AHC), CAPOS (cerebellar ataxia….)

Overall, a critical protein that affects many other proteins, and any mutation causing dysfunction may have large effects on health due to downstream effects

What different forms can secondary transporters be (3)?

1. Uniporters: passive transporters/facilitative diffusers (GLUT1)

2. Symporters: neurotransmitter transporters (EAAT/DAT/GlyT)

3. Antiporters: Na⁺/K⁺/Cl^- cotransporter (NKCC1)

What are neurotransmitter transporters and what is their role?

All NT transporters are secondary active transporters that are coupled to pre-existing ion gradients

There are two main families found on post- and pre-synaptic and glial cells:

1. Glutamate transporter family: Glutamate/aspartate

2. Neurotransmitter Sodium Symporter (NSS) Family: GABA, glycine, dopamine, NA, serotonin

Bacterial homologues of both fmailies have been crystallised and used as models - more recently, mammalian structures are available

Role: To clear NTs from synaptic cleft (important in brain)

What are EAAT transporters?

Excitatory Amino Acid Transporters (EAAT) are human plasma membrane glutamate transporters

• transport glutamate and aspartate with similar affinities (2 - 20 µM)

• 5 human subtypes (EAAT1 - 5)

  • EAAT1, 2 are found on glial cells and are widely expressed

  • EAAT3 - widely expressed on neurons

  • EAAT4 - neurons in cerebellum

  • EAAT5 - neurons in retina

• share ~50-60% amino acid identity

• EAAT2 ~1% of total brain membrane protein

The concentrating capacity of EAATs is linked to (A).

It can maintain a (B - no.) gradient across the membrane and has a net transfer of (C).

A - stochiometry

B - 10⁶ fold

C - two positive charges

Describe the structure of the transporter homologue from Pyrococcus horikoshii (Glt_ph)

Trimer made up of 3 identical subunits, bowl-shaped structure

• each subunit is capable of transport

• 37% identity to human EAAT2

• is an Na⁺-dependent aspartate transporter

L-TBOA is a competitive inhibitor of…

glutamate

What are hairpins in transporter topology?

Hairpins are structural motifs where a single alpha-helix bends or breaks in the middle, forming a loop-like structure.

• Instead of spanning the membrane completely, each half-helix enters and exits on the same side of the membrane.

• often plays a critical role in forming the substrate and sodium binding sites within the transporter

What did the cysteine proximity assay find in EAAT1 and Glt_ph?

The cysteine proximity assay tests if two cysteine residues are close enough (~6–7 Å) to form a disulfide bond, indicating spatial proximity.

In EAAT1, a double cysteine mutant showed:

• Increased function with DTT (reduces disulphide bonds).

• Decreased function with CuPh (forms disulphide bonds).

• Suggests the two cysteines are near each other and that their bonding affects transporter function.

In GltPh (K55C/A634C):

• The mutant protein was expressed, purified, and treated with DTT or CuPh.

• SDS-PAGE was used to detect disulphide bond formation.

• Confirms cysteine proximity biochemically.

What is the elevator mechanism of transport?

The elevator mechanism of transport is when part of a transporter protein (the "transport domain") moves like an elevator through the membrane, carrying the substrate from one side to the other, while the rest of the protein (the "scaffold domain") stays still.

• allows substrate to be moved across the membrane in a controlled, stepwise way

What is the mechanism of Glt_ph?

Glt_ph is the first elevator transporter

1. Binding: Sodium ions and substrate (like aspartate) bind to the transport domain on the outside of the membrane.

2. Elevator movement: The transport domain slides down through the membrane like an elevator, carrying the bound substrate to the inside.

3. Release: Substrate and sodium are released inside the cell.

4. Reset: The empty transport domain moves back up to the original position to start another cycle.

In which conformational states can Glt_ph exist (4)?

1. Outward-Facing State (OFS)

   • Substrate and 3 Na⁺ ions bind to the transport domain.

   • Binding site is exposed to the extracellular space.

2. Occluded State

   • Substrate is trapped inside; binding site is closed off from both sides.

   • Prevents premature release or leak.

3. Inward-Facing State (IFS)

   • Transport domain moves like an elevator through the membrane.

   • Binding site now faces the cytoplasm; substrate and ions are released.

4. Cl⁻ Conducting State

   • Separate from substrate transport; acts like a channel.

   • Triggered during intermediate conformations (likely between iOFS and IFS).

   • Allows passive Cl⁻ flux - uncoupled from substrate or Na⁺ movement.

   • Formed at the domain interface, creating a hydrophilic pathway.

   • Helps with charge balance and membrane potential regulation.

Excitotoxicity is the link between (A) and (B)

A - Na⁺/K⁺-ATPase

B - EAATs

Glucose transporter GluT1 belong to (transporter family)

Major Facilitator Superfamily (MFS) of secondary active transportes

• largest of all transporter families

• can be uniporters, symporters, or antiporters

Features of glucose transporters (3)

Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose across the plasma membrane

• through facilitated diffusion (uniporter) OR sodium-dependence

• GLUT (SLC2A) family is a protein family found in most mammal cells

  • 14 GLUTs are encoded by human genome

• Sodium-dependent glucose cotransporters (SGLT) are found in small intestine (SGLT1) and proximal tubule of nephron (SGLT1/2)

  • contributes to renal glucose reabsorption in kidney

  • if plasma glucose is too hh, glucose passes into urine because SGLTs are saturated with filtered glucose

Describe the structure of GLUT1

• 12 TMD (2 × 6 bundle)

• inward open conformation

• N- and C- domains connected by intracellular helical bundle (ICH)

  • mostly unique to sugar transporters

  • latch that tightens the intracellular gate

What is the mechanism of GLUT1?

Conformations:

• Outward-open (predicted, not captured)

• Ligand-bound, occluded (predicted from XylE)

• Inward-open (captured for GLUT1)

• Ligand-free, occluded (predicted from XylE)

Alternating Access Model: Substrate-binding site switches between outside and inside of the membrane.

ICH Domain: Latch that strengthens the intracellular gate in the outward-facing state.

Extracellular Gate: Formed by residues from TM1, TM4, TM7, controlling access to the binding site.

What is the neurotransmitter sodium symporter family?

NSS is a large family of NT transporters, includes:

• glycine, GABA, monoamines (dopamine, NA, and serotonin)

All human subtypes are coupled to co-transport of Na⁺ and Cl^-

They are targets for many therapies or drug abuse

• bacterial homologue from LeuT_Aa has been crystallised and is a model of the structure of all transporters in this family

What are the roles of GlyT1 and GlyT2 in inhibitory and excitatory neurotransmission?

Inhibitory

• GlyT2 is found exclusively on inhibitory synapses as glycine is an inhibitory NT

• Glycine receptors (GlyR) are ligand-gated Cl^- channels (not a transporter)

• GlyT2 clears glycine from synapse and uptakes it into pre-synaptic neuron

Excitatory

• glutamate is released and act on NMDAR and AMPAR receptors

  • these receptors also require glycine to bind as a co-agonist

  • glycine is transported by GlyT1 from astrocytes for excitatory NT

What could inhibition of GlyT1 and GlyT2 be used for?

GlyT1 inhibitors:

In schizophrenia, there is reduced NMDAR activity (NMDAR hypofunction hypothesis)

• inhibition of GlyT1 will elevate [Gly] at excitatory synapses and stimulate NMDAR

• Examples: sarcosine, NFPS (Merck, AstraZeneca, Organon)

GlyT2 inhibitors:

Inhibitory neurons are mostly found in the spinal cord. Inhibiting GlyT2 reduces excitatory transmission of pain signals from spinal cord up to the brain

• reduces perception of pain

• in spasticity, there is impaired glycinergic neurotransmission (GlyT2 inhibitors may be beneficial)

• ExampleS: ALX1393, N-Arachidonyl-Glycine, Oleoyl-L-Carnitine, other lipid inhibitors

What are the two subtypes of glycine transporters?

GlyT1 and GlyT2

• transports Na⁺ and Cl^-

• GlyT1 uses 2 Na⁺, GlyT2 uses 3 Na⁺ (stronger driving force → lower extracellular glycine)

What is the issue with current glycine transport inhibitors and how can it be addressed?

They are analgesic in chronic pain rodent models BUT have side effects - thought to be due to irreversible, competitive binding

→ without glycine, you can’t breathe, so you need some

• Alternative: bioactive lipids are “atypical” GlyT2 inhibitors

  • comprised of AA head and a monounsaturated tail

  • both the head and tail group are required for inhibition

  • are non-competitive

  • some transport of glycine still remains even at high concentrations of inhibitor

  • has potential to be useful in reducing pain signals with no serious side effects

Describe the binding of bioactive lipids to GlyT2

Bioactive lipids do not bind in the vestibule allosteric site

• act through extracellular allosteric site

• spontaneously binds to extracellular allosteric site in molecular dynamic simulations

Describe the ion coupling of catecholamine and monoamine transporters

DAT, NET, and SERT are all slightly different (do not need to know in detail)

Features of dDAT

dDAT is the Drosophila melanogaster dopamine transporter (dDAT)

• has 12 TM domains

• shot glass shape

• substrate buried

• co-transports 2 Na⁺ and 1 Cl^- ion

• Substrate and Na⁺ sites are similar in all members of the NSS family

Plasticity/flexibility confers (A) and determines (B)

A - versatile recognition

B - transport

Describe the CCC family

CCC refers to the Cation Chloride Cotransporter family (SLC12)

• moves Cl^- in and out of cells coupled to cations (Na⁺ and/or K⁺)

• uses Na⁺ and K⁺ gradients established by the Na⁺/K⁺-ATPase

Has many different types (only first two to know in detail):

• Na⁺-K⁺-2Cl^- co-transporters (NKCCs, SLC12A1-A2)

• Na⁺-Cl^- co-transporters (NCC, SLC12A3)

• K⁺-Cl^- co-transporters (KCCs, SLC12A4-A7)

• polyamine transporter (CCC9, SLC12A8)

• CCC interacting protein (CIP1, SLC12A9)

What is NKCC1 and its role?

NKCC1 is Na⁺/K⁺/Cl^- co-transporter 1

• responsible for cell volume regulation (water), chloride homeostasis, neuronal excitability

• As the immature nervous system develops into the mature nervous system, there is a shift from depolarising to hyperpolarising GABA_A receptor-mediated Cl^- currents takes place during neuronal development

  • switches direction that Cl^- moves

  • opposite effect following epilepsy and trauma

• In cortical and hippocampal neurons, NHCC1 mediates Cl^- uptake, while KCC2 extrudes Cl^-

• The energy for both of these electrically neutral ion-transport processes is derived from Na⁺-K⁺ ATPase

How was human NKCC1 structure determined?

By single-particle cryo-EM

Describe the structure of NKCC1

In an occluded and inward-open state

Membrane protein fold is highly linked to…

Mechanism (e.g. rocker switch, rocking bundle, elevator)

What is the main role of LGICs?

Fast neurotransmission

• NTs diffuse across synapse and bind to LGICs to trigger channel openings → ion fluxes (change ion gradients across cell membrane)

Identify the two main classes of LGICs

1. Pentemeric LGIC Superfamily (aka Cys-loop Receptors, Nicotinoid Receptors)

   • nACh receptors (excitatory)

   • 5-HT3 Receptors (excitatory)

   • GABAA Receptors (inhibitory)

   • Strychnine-sensitive Glycine Receptors (inhibitory)

2. Tetrameric Excitatory Ionotropic Glutamate Receptors

   • AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)

   • Kainate

   • NMDA (N-methyl-D-aspartate)

There are also ATP receptors (not covered in lecture)

Describe the structure of pentameric LGICs

• TM proteins consisting of 5 subunits

  • each subunit has an extracellular (blue in diagram) and transmembrane (yellow in diagram) domain

Describe the binding, gating, and activation of LGICs?

• M2 pore lining helices contain charged resides that confer ion selectivity

• In closed conformation, α helices bend towards centre of pore → leads to narrow region that is too narrow and too hydrophobic to allow ion passage

• Pore opening occurs via rotation and tilting of the pore lining helices

Identify 6 drug modulations of GABA_A receptors

1. Bicuculline: competitive antagonist of most GABA_A receptors

2. Benzodiazepines (diazepam, temazepam): enhances actions of GABA_A

3. Barbituates: may act alone or enhance actions of GABA_A

4. Ethanol: enhances actions of GABA → prolongs open time of channel, binds within TM domain

5. General Anaesthetics (e.g. propofol): enhances actions of GABA, may directly stimulate receptors at high conc., bing at interface between α and β subunits within TM region of channel

6. Neurosteroids (e.g. allopregnanolone): promotes channel opening on receptors containing α; d subunit is more sensitive

What is the MOA of benzodiazepines and pentobarnitone?

BZ acts selectively on GABA_AR that contain α1, α2, α3, or α5 subuntis, along with a γ subunit

• BZ binds at the interface of α and γ subunits (distinct from GABA binding site)

• do not activate the receptor by themselves but enhance GABA’s inhibitory effects by increasing channel opening frequency

• relatively safe

PB acts on all GABA_AR regardless of subunit composition

• increases the duration of chloride channel opening in response to GABA

• At high concentrations, PB can directly activate GABA_ARs even in the absence of GABA, which contributes to its sedative and anticonvulsant effects

• can be lethal

Describe the MOA of propofol on LGIC

Propofol is a general anaesthetic that binds to a distinct allosteric site (not BZ or GABA) → enhances the effect of GABA by increasing the duration of Cl^- channel opening

• short-acting drug

Describe drug modulation of glycine receptors

Strychnine: antagonist poison that causes paralysis

• blocks GlyRs → stops inhibitory neurotransmission → causes muscle contraction

• glycine and strychnine bind at the subunit interface

Ivermectin: allosteric enhancer

• irreversible agonist of GlyR → opens permanently

• binds in TM domains of GlyR

What are the subtypes of ionotropic glutamate receptors (2)?

1. NMDA-R: requires glutamate and glycine for activation

2. AMPA-R: requires only glutamate for activation

Distinguish between the electrical properties of AMPA and NMDA receptors

AMPA-R

• fast activation and desensitisation (inactivation)

• most selective for Na⁺

• some allow Ca²⁺ (dependent on RNA editing)

• widely expressed

NMDA-R

• slow activation and desensitisation

• allow Na⁺ and Ca²⁺ permeation

• voltage-dependent Mg²⁺ block

• widely expressed

What is the voltage-dependent block mechanism of NMDAR by Mg²⁺?

At resting membrane potential, the channel pore of the NMDA receptor is blocked by Mg²⁺ ions

• prevents flow of cations even when the receptor is activated by glutamate and co-agonist glycine

When AMPA-R is activated, depolarisation will cause Mg²⁺ to be expelled

• allows ions to flow through

The Mg²⁺ block ensures that NMDARs only become active when two conditions are met:

1. Presynaptic glutamate release (ligand binding)

2. Postsynaptic depolarisation (Mg²⁺ removal)

Describe the structure of excitatory glutamate receptors

• 4 subunits; each has 4 TM domains

• has really large extracellular domains

How does the structure of excitatory glutamate receptors aid in its function?

Ligand-binding domain (LBD) is termed a “venus flytrap” domain

• when ligand binds, LBD engulfs the ligand

  • causes various domains to rotate and to move → pulls gate apart to allow channel opening

There is a region in the selectivity filter of the pore that can also undergo change

• residues in TM2 control cation permeability (RNA editing)

• flip/flop region yields 2 splice variants for each subunit

How can AMPA-R be affected by RNA editing?

GluRA1 pre-mRNA undergoes RNA editing, where an adenosine is changed to inosine (A-to-I)

• changes codon at pos. 607 → changes Q to R residue

• editing alters Ca²⁺ permeability