Lecture 1 - Channel Gating and Modulation

What Determines the Macroscopic (Whole Cell) Current Recorded in Electrophysiology (Patch Clamp Experiment)?

• P = channel open probability

• i = single-channel (unitary) conductance (biophysical gating properties)

• N = number of functional channels in the membrane

  • Whole-cell current (I) = P × i × N

• These properties are important in the regulation of whole cell currents

What Does The Patch Clamp Experiemt Measure in a Whole Cell Mode

• Patch clamp in whole-cell mode measures macroscopic currents (e.g., inward Na⁺ currents, outward K⁺ currents), which arise from the summed activity of many ion channels.

What Are the Biophysical Properties of Ion Channels, and What Do They Determine

• Channel open probability (P) and unitary conductance (i)

  • They determine gating and ion permeation.

• These properties are intrinsic to the pore-forming subunits, but can be modulated by accessory subunits or signalling/regulatory proteins.

Based on the Equation I = P · i · N, How Can Ion Channels Be Targeted Therapeutically? 

• Channel activity

  • Gating and ion permeation

  • Can be modulated directly or indirectly

• Channel density (N)

  • By altering transcription, trafficking, or membrane expression

What is the Structure of the Kv Alpha Subunit?

• 4 α-subunits assemble to form one channel

  • Each α-subunit has 6 transmembrane segments (S1–S6)

• S1–S4 = Voltage-sensing domain

  • S4 contains positively charged residues → acts as the voltage sensor

• S5–S6 = Pore domain

  • S5–S6 + pore loop forms the ion-conducting pore

  • S6 forms the activation/inactivation gate 
    • Structural variability across genes → functional variability between channel subtypes

• N-terminal Tail: facilitates fast inactivation

• C-terminus Tail: faciliates slow inactivation

What Are the Two Main Types of (Kv) Channel Inactivation?

• N-type inactivation (fast):

  • Mediated by N-terminal “inactivation particle” that blocks the pore

  • “Ball-and-chain” mechanism

    • differs between different types of channel 

• C-type inactivation (slow):

  • Conformational change/constriction at the pore

  • Mediated by rearrangements involving S6 alpha helices/pore region

What is Electromechanical Coupling in Kv Channels>

• A change in membrane voltage causes the S4 voltage-sensor to move outward

• S4 movement interacts stepwise with negative residues in S1–S3 (Gating Charge Transfer Centre) 

• This movement is mechanically linked to the opening of the S6 activation gate

• Channel opening is followed by inactivation (fast N-type or slow C-type)

What is the Effect of Electromechanical Coupling Achieve in Kv Channels?

• It links voltage sensing (S4’s pairwise movement and interation with negative charges on S1-S3) to opening of the pore (via S4–S5 linker displacement)

• Ensures that changes in membrane potential directly regulate ion flux

• Triggering of pore opening automatically initiates inactivation processes (N-type or C-type), ensuring controlled, time-limited K⁺ currents

• This coordinated activation + inactivation shapes action potential repolarisation and firing patterns

What Are The 3 Main States That Ion Channels Exist In?

• Resting (close) state: Membrane not sufficiently depolarised → Membrane potential below threshold → channel can’t open

• Open (activated) state: Membrane sufficiently depolarised → electromechanical coupling → ion influx/efflux 

• Inactivation state: Prolonged depolarisation causes channel to close → via N-type inactivation particle or C-type pore constriction.

What Are State-Dependent Blockers?

• Many ion channel–targeting drugs in clinical use (e.g., L-type Ca²⁺ antagonists, local anaesthetics) preferentially bind to specific channel states, particularly the inactivated state, to elicit block.

• Because the proportion of channels in each state depends on membrane voltage  and depolarisation, drug binding becomes state-dependent = voltage-dependent

• Result: Drugs are most effective in tissues that are frequently depolarised (more channels in the inactivated state).

Name 3 examples of L-type Ca²⁺ channel blockers, their mechanism of block and clinical use.

• Dihydropyridines – anti-hypertensive

• Phenylalkylamines – anti-arrhythmic

• Benzothiazepines – anti-arrhythmic & anti-hypertensive

• Mechanism: Exhibit use- and/or voltage-dependent block – preferentially target inactivated channels, and ‘locks’ them in the inactive state.

  • Channels are most effectively blocked when frequently depolarised

How Does Drug Binding Affect Ion Channel Gating and Ion Permeation 

• Drugs (DHPs, PAAs, BTZs) bind non-competitively to distinct but overlapping sites in the pore region of transmembrane domains III & IV.

• Binding alters protein conformation, affecting:

  • Channel gating (opening/closing)

  • Ion permeation (ion flow through pore)

• Specific amino acids in the binding pocket, involved in drug binding, influence electrophysiological properties (voltage-dependent inactivation, ion conductance).

• Result: state-dependent channel block (preferentially blocks inactivated channels).

How Do Dihydropyridines (DHPs) Achieve Voltage Dependent Block of L-Type Ca2+ Channels

• They preferentially bind inactivated channels → block is strongly voltage-dependent.

• Block is enhanced with stronger depolarisation: more channels enter the inactivated state → greater block.

How Do PAAs and BDZ Achieve Use/Frequency Dependent Block? 

• Block builds over time with repeated depolarisations (high AP frequency).

• More frequent depolarisations → more channels open and enter the inactivated state → block accumulates over time.

How Does The PAA (phenylalkylamines) Verapamil Achieve Use/Frequency-dependent Block?

• The anti-tachycardic preferentially binds open channels, then locks them in the inactivated state.

• * *

• Targets channels with high electrical activity → reduces Ca²⁺ influx → slows heart rate.

• Key points:

  • Needs a channel open to reach the binding site

  • Binds & “freezes” inactivated channels

  • Higher AP frequency → high probability of block (­­increased potency)

How Do Local Anaesthetics (e.g. Lidocaine, Prilocaine) Block Nav Channels?

• Bind to S6 α-helical segments in domains I, III & IV of the pore region.

• Use-dependent block: preferentially binds inactivated channels → “lock” channels in inactive state.

  • Pan-specific: target all Nav subtypes.

How does the carbamazepine block Nav channels and why is it clinically useful?

• Binds to residues overlapping the local anaesthetic site in Nav channels.

• Use-dependent blocker → preferentially targets overactive channels.

• Clinically useful in pain, hyperalgesia, and epilepsy, where neurons have heightened activity.

  • anti-epileptic and anti-nociceptive

Why is it difficult to achieve state-dependent block when targeting the channel pore?

• High sequence homology between channel subtypes → drugs binding the pore often lack selectivity.

• Highly conserved pore sequences make it difficult to develop subtype-specific blockers.

• Targeting the pore alone can cause off-target effects in other tissues expressing similar channels.

What is the Alternative Approach to Achieveing State Dependent Block of Channels?

• VSDs exhibit greater variability between channel subtypes, allowing selective targeting.

• Drugs like ICA-121431 interact with S2 & S3 of VSD IV in Nav1.3/1.1 → confers high subtype selectivity.

• Example: 3 key amino acids in the VSD confer 1000-fold sensitivity to icogenin compounds.

• VSD-targeted modulators enable subtype-specific block → useful in pain (Nav1.3) or epilepsy (Nav1.1).

How Does the VSD Modulator ICA Inhibit Nav1.2?

• It causes almost complete block at depolarised potentials, minimal block at hyperpolarised potentials → voltage-dependent (state-dependent) block.

• Targeting VSD IV indirectly affects gating and ion permeation.

• Clinically relevant for pain treatment.

How Does The Kv Channel Opener NH29 Inhibit Electrical Excitability?

• Enhance activity/opening of inhibitory Kv channels → K⁺ efflux → reduces excitability.

• The diphenylamine carboxylate binds externally at the S1-S2-S4 interface of Kv7.2.

  • Stabilises interactions between S2 negative charges and S4 positive residues → promotes channel opening → K+ efflux

  • Effect on current: Increases current, slows activation, leftward shift in activation curve (greater K⁺ flow at the same voltage).

• Clinical relevance: Kv7.2 regulates cellular excitability → target for epilepsy therapy.

How Does VSD Modulators Achieve State Depdent Block Effects

• Voltage-sensing domains (VSDs) move up/down depending on the channel state (open, closed, inactivated).

• VSDs adopt distinct conformations that create distinct ‘allosteric’ sites for drug binding

• Drugs targeting VSDs can modulate gating and ion permeation in a state-dependent manner.

What is Significance of mutation sin the VSDs of Nav and Cav Channels

• VSDs are critical for electromechanical coupling and channel opening.

• Mutations can cause channelopathies (e.g., epilepsy, pain syndromes, dilated cardiomyopathy).

• Thus, they are important therapeutic targets for modulating channel function.

How Can Ion Channel Activity be Modulated

• State-dependent block: Target the channel pore or allosterically modulate gating/ion permeation via the voltage-sensing domain (due to its variable amino acid sequence across subtypes).

• Regulatory modulation: Signalling molecules or regulatory/accessory subunits can interact with the pore-forming subunit and modify gating, ion permeation, and trafficking.

How Does Structural Diversity in Ion Channels Arise

• Differences in pore-forming α-subunits generate functional diversity.

• Example: Kv channels have 12 different gene families encoding α-subunits, with multiple splice variants, allowing subtle differences and distinct physiological roles.

• Diversity is increased by auxiliary subunits, which modify basic channel properties  

How Do Auxillary Subunits Contirbute to Ion Channel Diveristy and Function?

• Subunits modify gating, ion permeation, and current, and promote trafficking to the cell surface.

• Examples:

  • Kvβ1-3 (KCNAB1-3): cytoplasmic protein assocaited with N-terminal tail, contributes to fast inactivation of Kv1 channels

  • minK (KCNE1): transmembrane protein

  • BKCA: transmembrane protein with large extracellular domain, interacts with extracellular proteins

  • MiRP1-3 (KCNE2-4), KCNE1L, KCHIP1-4 (KCNIP1-4)
    •

• Variability in auxiliary β-subunits allows cells to create channels that fulfill niche functional roles downstream of ion flux

What is The Structure of Voltage-Gated Ca Channels (Cav)?

• Composed of α1, β, and α2δ subunits.

  • High Voltage-Activated (HVA) channels: α1:β:α2δ in 1:1:1 stoichiometry

  • Low Voltage-Activated (LVA, T-type) channels: α1:(α2δ) in 1:1 stoichiometry

• There are 10 α1 subunits and 4 β subunits in total (depending on the subtype).

How Do Cav B-Subunits Affect The Trafficking of Voltage-Gated Ca Channels?

• Without β1, channels are trapped in the secretory pathway → minimal channel surface expression.

• Co-expression with β1b increases:

  • Surface expression of the channel

  • Whole-cell current (more channels + enhanced Ca²⁺ kinetics)

How Do Cav B Subunits Modulate Channel Gating and Current Kinetics?

• Co-expression shifts the current–voltage relationship left → more current at a given membrane potential.

• Channels become easier to open or remain open longer → affects voltage-dependent activation and gating.

How Does The Type of B-Subunit Influence Cav Channel Inactivation?

• Co-expression with different β subunits alters Ca²⁺ influx and marked difference in the shape of the current:

  • β1b, β3: marked inactivation

  • β2a, β4: little/no inactivation

• Choice of β subunit impacts downstream cellular function by controlling Ca²⁺ entry.

How do β1 and β2 subunits differentially modulate cardiac L-type Cav1.2 channel inactivation?

• β2: slow/no inactivation → inactivation particle tethered via palmitoylation of the N-terminal cysteine residues → inactivation particle cannot act

• β1: fast inactivation → inactivation particle free to close channel normally

Why do β1 and β2 subunits produce different inactivation kinetics?

• The functional core (SH3 + GK domains) conserved in the subunits

• Hypervariable N- and C-terminal regions differ

• β2 has shorter N-terminal + 2 cysteines residues which undergo palmitoylation → B2 tethered to membrane → prevents inactivation

• β1 cannot be palmitoylated → normal (fast)  inactivation

What is the Effect of Palmitoylation of the N-Terminal Cys Resiudes in the B2 Subunit?

• slow current inactivation of cardiac L-type Cav1.2 channels

• Inactivation particle not free to move and inactivate the channel

  • slow/ no inactivation over depolarising pulses

How does β subunit modulation of Cav1.2 affect cardiac action potentials?

• β2 → slow, sustained Ca²⁺ influx → supports long plateau phase of cardiac AP

• β1 → rapid inactivation → shorter Ca²⁺ influx

• Kv7.1 association with MinK accessory subunit → slow K⁺ efflux → repolarisation

• Proper subunit association is crucial for normal heart rhythm; dysregulation → arrhythmias

Why is differential β subunit modulation of Cav1.2 clinically important?

• Altered channel inactivation can affect cardiac excitability

• Dysregulated Ca²⁺ influx can cause arrhythmias and potentially sudden death

How do α2δ subunits modulate CaV channels?

• Increase surface expression of Cav α1 subunits → more channels at the membrane

• Enhance current and speed up activation/inactivation kinetics

• Hyperpolarise the voltage-dependence of activation → channels activate more easily

• Promote trafficking and stabilisation of channel complexes at the cell surface

• Shift current-voltage relationship → larger Ca²⁺ influx

  • Function similarly to beta subunits, with a less pronounced effect on trafficking or biophysical properties

What is the Functional Consequence of a Lack of α2δ subunits On CaV channels?

• Small current

• Slow channel activation

• Little to no channel inactivation

How Do Gabapentinoids Modulate Cav Channels?

• Target auxiliary α2δ-1 and α2δ-2 subunits, not the pore-forming α1 subunit

  • Used in epilepsy and chronic pain

• Disrupt trafficking and other α2δ-mediated properties → reduce Ca²⁺ channel function without directly blocking the pore

How Does PKA Modulate Cav2.1Channels in the Heart

• Regulatory proteins can modulate the gating and ion permeation of Cav1.2

• PKA phosphorylates Cav1.2 → increases channel open probability and number of channel openings

  • Increases current amplitude

• Result: ↑ Ca²⁺ current → ↑ force of cardiac contraction

• Critical for excitation-contraction coupling in the heart

How Does Alternative Splicing of Cav2.1 Channels Affect PKA Regulation

• Cav1.2 is widely expressed and has multiple splice variants (cardiac, smooth muscle, etc.) → different channe properties

• PKA phosphorylates Cav1.2 at a conserved site to modulate of channel activity

• Some splice variants lack the PKA consensus site → not regulated by PKA

• Only the full-length cardiac Cav1.2 α1 subunit is phosphorylated and modulated

What is the Role of AKAP (A-kinase anchoring protein)  in PKA’s Modulation of Cav2.1?

• α1 subunit: phosphorylated at the C-terminal tail by PKA

• β2 subunit: must also be phosphorylated for the full PKA modulation effect

• It is a scaffold protein anchored at cytoplasmic membrane and has regulatory and catalytic domains

  • It recruits PKA to Cav1.2 channel complex

  • Facilitates efficient protein-protein interaction between α1 and β2 subunits

  • Forms a mini signalling complex around individual channels for precise regulation

How do Cav1.2 splice variants influence channel function and regulation? 

• Splice variants of Cav1.2:

  • Determine physiological function

  • Affect whether the channel is modulated by PKA, PKC, etc.

  • Influence drug sensitivity and targeting

AKAP Scaffold:

• Protein tethered at membrane

• Promotes protein-protein interactions (PPIs) between PKA and Cav1.2/β2 subunits

• Forms a signaling complex for precise modulation

How Do G-Proteins Modulate Presynaptic Cav2 Channels?

• Gβγ binds Cav2 channels → inhibits current → slowed channel activation → small Ca²⁺ influx

• Voltage-dependence: Inhibition relieved by strong depolarisation (train of AP’s) → faster activation → facilitation of Ca²⁺ currents

  • Large depolarisation required for faster activation → bigger Ca2+ trigger for NT release

• Outcome: Reduces neurotransmitter release → decreases neuronal hyperexcitability

• Clinical relevance: Mechanism of opioid analgesia at presynaptic terminals

• Produces tonic inhibition of N-, P/Q-, R-type Ca²⁺ currents

How Are Cav Channels Regulated and What is the Significance of This?

• There are multiple regulatory pathways → CaV1.2 has numerous consensus sites for modulation by signalling molecules (e.g., kinases, G-proteins)

• This allows fine-tuning of channel activity to fulfil specific cellular functions