Lecture 2: Ion Channels As Theraputics -> Trafficking and Surface Expression

How Does Ion Channel Distribution Affect Excitable Cell Function

• Ion channel distribution defines the function of excitable like nerves and muscles

• Pore forming and auxiliary subunits differ across regions of the cell, affecting the frequency, size, shape, and pattern of action potentials.

• Electrical impulses code messages through excitable cells.

• VG ion channels are critical for normal excitable cell function and can also play roles in unexpected contexts (e.g., strongly metastatic cancer cells

How Does Gentic Diveristy Affect Ion Channel Function

• The genetic diversity of a channel underlies the structural diversity of a channel’s individual subunits and, as such, impacts their functional properties (gating, ion permeation)

• Alternative splicing of mRNA further increases functional diversity.

• Allows cells to tailor channel properties to specific physiological roles

How Does Genetic Diversity Manifest in High Voltage Activated Ca2+ Channels

• It posses

  • 10 different α1 subunits (10 genes)

  • 4 different β subunits (4 genes)

  • 4 different α2δ subunits (4 genes)

• Combinatorial possibilities: 10 × 4 × 4 = 160 “flavours” from genes, each with different proerties

• Alternative splicing (e.g., Cav1.2 α1 has 47 exons) can potentially create thousands of subtypes.

• Cells can precisely select subunit combinations for specific functions.

Why is regulating the surface density and localisation of ion channels crucial for cell function?

• Surface density & localisation of channels determine functional specificity in discrete cellular locations.

• Channels must reach the correct location to interact with modulatory proteins and perform their function.

• Diversity of subunit combinations allows cells to tailor channel properties to local demands.

How Do Ion Channels Regulate Surface Density and Localisation of Ion Channels

• This is medated by:

  • Coordinated synthesis, assembly, and trafficking of channels.

  • Ability to sense cellular need → initiate transcription of required channels → traffic to specific locale

How Are CavB Subunits Differentially Locasilsed in Hippocampal Neurons

• β2a & β4: Punctate distribution → clustered at the cell surface

• β1b & β3: Diffuse distribution → throughout neurites and cell body

• Synaptic association:

  • β4: Colocalises with synaptic proteins (e.g., synapsin, synaptobrevin) → channels likely involved in neurotransmitter (NT) release

  • β2a: Not colocalised → less involved in NT release

• Role: Subunit localisation modulates channel activity and trafficking

How does Cavβ subunit type affect calcium influx and neurotransmitter release in hippocampal neurons?

• β4:

  • Little inactivation over a depolarising pulse → sustained Ca²⁺ influx → larger NT release

  • Channels associated with synaptic proteins → efficient NT release

• β3:

  • Rapid inactivation → brief Ca²⁺ influx → short NT release

• Differential Cavβ subunit distribution tailors Ca²⁺ signals to meet synaptic needs

How Are Ion Channels Trafficked to the Plasma Membrane in the Secretory Pathway

• Synthesis in Nuclei and cytoplasm: Gene transcription → translation → immature protein

• Transport to ER for Quality Control:

  • Ensures proper folding & assembly

  • Determines whether protein progresses or is degraded

  • Examples:

    • Cleavage of α2δ Cav subunits

    • Assembly of 4α + 4β for Kv channels (oligomeric structure)

• Transport to Golgi & Trans-Golgi Network For:

  • Post-translational modifications: phosphorylation, palmitoylation, glycosylation

  • Modifications affect folding, functional properties, and surface expression

How Is Ion Channel Expression Regulated Upon Exit From The Secretory Pathway

• Packaged into vesicles → transported to plasma membrane

• Surface dwell time: Finite and is influenced by associated stabilising proteins

• Fate after insertion: Internalisation, recycling, or degradation

• Balance of trafficking: Forward (cytoplasm → surface) vs backward (internalisation → degradation)

• Determines the number of functional channels and cell excitability

What is The Role of Accessory (Auxiliary) Subunits in Ion Channel Assembly and Trafficking? 

• Modulate channel activity

• Promote forward trafficking of correctly folded pore-forming (α) subunits to the plasma membrane

• Stabilise channels once at the cell surface

• Assembly occurs in the ER alongside quality control

How does the ER ensure that only correctly folded and assembled ion channels are trafficked to the plasma membrane

• ER retention signals are present in the proteins and prevent misfolded or incomplete proteins from exiting the ER

• Correct assembly masks ER retention motifs:

  • HVA Cav1/Cav2 channels: β subunit binds intracellular I–II loop → covers ER retention signal → mature channel exits ER and goes to the plasma membrane

  • Kir/KTP channels: Octamer formation (4 α + 4 auxiliary subunits) masks retention motifs → allows trafficking to the surface

How does the ER detect misfolded or misassembled Kv channels and ensure only functional tetramers reach the plasma membrane?

• Kv channels require tetrameric assembly (4 α subunits) to function

• Conserved Thr on S1–S2 extracellular loop bridges VSD (S1–S4) and pore domain (S5–P–S6) of adjacent subunits, which is required to stabilise the tetramer (forms a bridge) 

• Wild-type Thr → proper assembly → robust outward current

• Mutation (Thr → Ala) → channel trapped in ER → no surface expression, no outward current seen 

• ER ensures that only functionally coupled subunits reach the cell surface

• The mechanism of ER recognition of bridged subunits is not fully understood

How does mis-trafficking of ion channels contribute to Disease

• Mis-trafficking of ion channels can cause dysfunctional cell excitability, e.g., cardiac channelopathies → arrhythmias

• Implicated in diseases like Long QT Syndrome (LQTS)

• Mis-trafficking can involve pore-forming and modulatory subunit

What Are the Clinical Features of The LQT Syndrome, Romano Ward?

• Increased QT interval on ECG

• Higher risk of torsades de pointes → ventricular fibrillation

  • Can lead to sudden cardiac death

• Associated with inherited mutations in cardiac ion channel genes in both the pore-forming and modulatory subunits

How does LQTS1 (Romano-Ward Syndrome) affect Kv7.1 (KCNQ1) channels?

• Most common subtype of R-W LQTS

• Mutations in KCNQ1 (Kv7.1) → slow repolarising K⁺ current (IKs) in the heart

• Effects of mutations:

  • Disrupt tetramer assembly → channel mis-trafficking

  • Alters association with KCNE1 (minK) accessory subunit

  • Result: reduced functional K⁺ channels at the membrane → prolonged repolarisation

How do mutations in Kv7.1/KCNQ1 channels cause Romano-Ward Syndrome (LQTS1)?

• Key amino acids in the intracellular N-terminal tail of the Kv7.1 (KCNQ1) α-subunit are critical for channel trafficking.

• These key amino acids can be mutated out →

• Example: Tyr → Cys mutation

  • Causes the channel to be retained in the ER

  • Result: little to no K⁺ current at the membrane

  • Wild-type channels show robust outward K⁺ current

What Key Post-Translational Modifications Occur As A Protein Moves Through the Golgi?

• Glycoslyation

• Ubqutination

• Phosphoylation

• SUMOylation

What is Glycosylation?

• The addition of sugar chains (glycans) to proteins.

  • Role in ER: Ensures proper folding during quality control.

  • Role in Golgi (cis/medial/trans): Final processing impacts functional properties of proteins/channels.

What is Ubiqutination

• The process of tagging a protein for degradation by the proteasome 

• It can alter channel activity 

• It is important in:

  • Quality Control in the ER

  • Regulation at cell surface, e.g. channel density – decreases n.o channels at the surface

  • Regulating protein localisation (nuclear cytoplasmic shuttling or targeting to  specific sub-cellular compartments)

How Does Phosphorylation Affect Ion Channel Surface Expression?

• In the cis-Golgi it dictates final destination of proteins

• In the cytoplasm and at the cell surface, it acts to regulate surface expression, &/or activity.

What is SUMOylation?

• It is the addition of Small Ubiquitin-like Modifier  protein to a protein

• It acts to stabilise and modify protein-protein interactions at the cell surface to regulate surface channel expression

• Effect:

  • Impacts protein stability by promoting degradation or stabilisation

  • Regulates protein localisation (nuclear cytoplasmic shuttling or targeting to specific sub-cellular compartments

  • Assessed to explore changes in ion channel expression that is associated with chronic and neuropathic pain

How Is Nav1.7 Channel Surface Expression Regulated in Chronic and Neuropathic Pain? 

• Channel expression is regulated by SUMO and CRMP2

• Upregulation during pain → increased pain experienced due to SUMOylation of CRP2 in sensory neurons

  • SUMOylated CRMP2 binds to Nav1.7, increasing its trafficking and functional expression 

• DeSUMOylation of CRMP2 recruits an endocytic complex  (Ub, Numb, Eps15, Nedd4-2), which causes endocytosis and internalisation of Na_v1.7 and reduces surface expression → reduces pain

  • targeted with novel channels

How is Cav2.2 and Cav3.2 Surface Expression Regulated in Pain?

• Channels play a key role in pain transmission through the regulation of NT release between the 1° and 2° afferent neurons in the spinal cord

  • SUMOylation of CRMP2 (via Cdk5-dependent phosphorylation) → binds Cav 2.2 channel → increases Cav2.2 surface expression in heterologous systems → may increase pain.

    • Effect in sensory neurons: still unclear.

  • DeSUMOylation by USP5 promotes Cav3.2 trafficking & surface expression → observed in sensory neurons → increases channel activity.

  • Knockdown of USP5 → increased ubiquitination of Cav3.2 → decreased channel activity → analgesia.

How is the surface expression of cardiac L-type channels regulated via the β2 subunit?

• Auxiliary β2 subunit stabilises Cav1.2 channels at the plasma membrane.

• Mechanism: Akt phosphorylates the β2 C-terminus → enables interaction with TID (Tail Interaction Domain) in SH3 → structural rearrangement stabilises channel at the cell surface→ more channels at the cell surface.

• Therapeutic insight: Phospho-mimetic peptide (R7W-MP) mimics β2 phosphorylation → stabilises channels → potential to modulate cardiac excitability.

• Targeting trafficking/stabilisation of auxiliary subunits can alter channel expression levels!

How Does B2 Phosphorylation Regulate Cav1.2 Trafficking To The Cell Surface?

• Channel subunits (α1, β, α2δ) assemble into a complex in the ER → traffic to the plasma membrane.

• Unphosphorylated β2: binds dynamin → triggers reverse trafficking & degradation → fewer channels at surface.

• Phosphorylated β2 (at AKT consensus site): prevents dynamin binding → reduces reverse trafficking → stabilises channels → more at cell surface → improves cardiac function (e.g., in diabetes).

How does cytoplasmic β2 regulate Cav1.2 transcription in the nucleus?

• Some β2 subunits are not complexed with α1 and can linger in cytoplasm, where they behave promiscuously and bind to KIR-GEM-GTPase and translocate to the nucleus and associate with HP1γ (transcriptional regulator), which represses CACNA1C gene transcription, which encodes L-type Cav pore-forming subunit, thus reducing L-type channel production.

• Phosphorylated β2 blocks this pathway → no nuclear translocation → normal Cav1.2 transcription.

How Can Phosphomimetic Peptide (R7W-MP) Be Used To Stabilise Cav1.2 ?

• Mechanism: Targets Cavβ2 auxiliary subunit → stabilises Cav1.2 at the cell surface.

• Mouse model (Streptozotocin-induced diabetes):

  • ↓ Cav1.2 surface density → impaired ventricular contractility

  • R7W-MP treatment: restores Cav1.2 levels in diabetic mice and normalises ventricular systolic function

• Translational relevance:

  • Human left ventricular biopsies show a correlation between Cav1.2 levels and Akt medated Cavβ2 phosphorylation

  • Represents a novel approach to modulate Cav1.2 expression and function

• Proof-of-concept: Potential therapeutic tool for cardiac conditions associated with Cav1.2 dysfunction

What is the role of α2δ subunits in CaV channel function at synapses

• α2δ subunits stabilise CaV channels at the synapse.

• Example: Drosophila α2δ (straitjacket, stj), mammalian equivalent exists.

• Loss of α2δ (stj-null):

  • Reduces surface expression of CaV channels

  • Causes severe neurotransmitter release defects (flies cannot move their wings)

What is the Role of α2δ subunits in CaV channel function in Hippocampal Neurons

• α2δ subunits promote the expression of Cav2.2 channels pre-synatpically to promote neurotransmitter release

• Loss of α2δ subunits or disruption in their function (e.g. with GBP treatment) can reduce channel densitry and NT release

Why is the spatial distribution of ion channels within cells important?

• Channels must be in the correct cellular location to function properly.

• Proper localisation ensures interaction with the correct regulatory proteins, enabling channels to fulfil their specific roles

How do scaffold proteins like PSD-95 influence ion channel localisation and function

• Kv1.4 channels and PSD-95 are diffusely distributed when expressed alone.

• Co-expression leads to clustering of channels with scaffold proteins at the cell surface.

• These clusters form signalling platforms:

  • Allow high-density channels together

  • Facilitate their modulation by signalling proteins

  • Stabilise channels at the cell surface for functional activity

How Does PSD-95 Stabilise Ion Channels At The Cell Surface?

• PSD-95 scaffolds pull ion channels together into high-density clusters.

• It is composed of functional domains:

  • PDZ domains: Bind channels and recruit signalling molecules to regulate channel activity at cell surface

  • GK (Guanylate Kinase) domain: Supports cytoskeletal docking and has residues that are palmytolated

  • Palmitoylation sites: Tether scaffold to membrane.

• Scaffolds link and tether channels to the membrane & cytoskeleton, prolonging their surface residence.

• Macromolecular signalling complexes can cluster and multimerise, forming a large regulatory molecules to coordinate channel activity

• Without PSD-95, channels are rapidly removed from the membrane, reducing functional activity.

How does α2δ target Cav channels to lipid rafts, and why is this important?

• α2δ defines the Cav channel location by targeting them to lipid rafts.

• Lipid rafts are highly ordered regions of the plasma membrane rich in cholesterol & sphingolipids, which concentrate signalling molecules (G-proteins, PKs), and modulate ion channel function.

• α2δ has a similar structure to integrins → regulates cell adhesion, ECM cell-cell contact, and transmits bidirectional signals.

• Function:

  • α2δ partitions GBP-sensitive α2δ-1/2 + Cav2.x channels into lipid rafts.

  • Crucial for NT release and CNS synaptogenesis (via thrombospondin interaction).

• Disruption of lipid rafts changes Cav2.1/2.2 biophysical properties and channel function

What does sucrose fractionation reveal about α2δ-1 and Cav2.2 α1/B1 complexes and their association with lipid rafts?

• Purpose: Isolate and assess protein localisation to lipid rafts.

• Method: Sucrose gradient fractionation → separates proteins by buoyancy:

  • Raft-associated proteins: float to 5–30% sucrose (top) → buoyant layer. Caveolin used as a raft marker.

  • Non-raft proteins: remain in high-concentration sucrose (bottom).

• Results:

  • α2δ-1 and Cav2.2 α1 expressed separately → non-raft layer.

  • α2δ-1 + Cav2.2 α1 co-expressed → some protein detected in buoyant lipid raft layer, indicating assembly and entery into the secretory pathway.

How does localisation in lipid rafts vs non-raft areas affect CaV channel function?

• Lipid rafts bring together high concentration of signalling/regulatory proteins → channels here experience different modulation compared to non-raft areas 

• Non-raft areas may have fewer or different signalling proteins → distinct regulation.

• Functional implication:

  • Ca²⁺ influx through the same channel can trigger different downstream pathways depending on its localisation in a raft or non-raft area, due to the presence of different signalling molecules

  • Adds a layer of fine-tuning, allowing cells to differentially modulate ion channels based on location.