Voltage-Gated Potassium Channels Comprehensive Notes

Voltage-Gated Potassium Channels (Part 1)

Introduction to Voltage-Gated Potassium Channels

• The lecture focuses on the structure and function of voltage-gated potassium channels (KV channels).

• Topics include:

  • Classification of different potassium channels, including the role of KV channels in the action potential.

  • KV channel structure, voltage sensitivity, and ion selectivity.

  • Inactivation and phosphorylation of KV channels, including toxins affecting KV channel function.

The Role of Ion Channels in Neuronal Function

• Neurons rely on ion channels for their electrical properties.

• Ion channels are essential for generating electricity in neurons.

  • Membrane potential (Vm) is the voltage across the membrane, measured in millivolts (mV).

  • At rest, $Vm = –65 mV$.

  • This potential arises from differences in electrical charge across the membrane.

  • The inside of a cell is more negative relative to the outside.

  • A negative charge inside a neuron is crucial for a functioning nervous system.

Resting Membrane Potential (RMP)

• Ion channels are essential for maintaining the RMP.

• The RMP is a balance between the equilibrium potentials for potassium (K+) and sodium (Na+), highly dependent on the membrane's permeability to each ion.

  • $K^+<em>{in} = 100 mM$, $K^+</em>{out} = 5 mM$, $E_K = –80 mV$, Permeability = High (40), Some K+ leaks out

  • $Na^+<em>{in} = 15 mM$, $Na^+</em>{out} = 150 mM$, $E_{Na} = +62 mV$, Permeability = Very Low (1), Some Na+ leaks in

  • Using the Goldman equation, the resting membrane potential is: $Vm = –65 mV$

Ion Channels and Neuronal Communication

• Ion channels enable neurons to communicate with each other.

Action Potential Phases

• The action potential consists of five phases:

  1. Resting State: The membrane is at the RMP (–65 mV).

  2. Rising Phase: A rapid depolarization of Vm.

  3. Overshoot: The inside of the neuron becomes positive relative to the outside.

  4. Falling Phase: Rapid repolarization of Vm.

  5. Undershoot: The inside of the neuron is more negative than at rest (hyperpolarized).

• Sodium (Na+) channels underlie the rising phase of the action potential.

  • At threshold, voltage-gated sodium channels open, allowing sodium to flow into the neuron, depolarizing Vm.

• Potassium (K+) channels underlie the falling phase.

  • As voltage-gated sodium channels inactivate during the overshoot, voltage-gated potassium channels open, allowing potassium to leave the neuron, repolarizing Vm.

Channels Mediating Action Potential Phases

• Synaptic potentials depolarize the neuron to threshold, activating $Na_V$ channels at the hillock, causing sodium to rush into the neuron.

• $K<em>V$ channels begin to open at the overshoot; at the same time, $Na</em>V$ channels inactivate, and Vm is nearly at $E_{Na}$.

• Potassium flows out of the neuron through the open $K<em>V$ channels, repolarizing the membrane potential; $Na</em>V$ channels are still inactivated.

• $K<em>V$ channels take longer to close, causing Vm to reach $E</em>K$, producing the undershoot.

• During the undershoot, the Vm is refractory (absolute then relative).

• In the relative refractory period, $Na_V$ channels de-inactivate, making it possible to trigger another action potential.

Potassium Channel Families

• Potassium channels enable K+ to flow across the membrane with great selectivity.

• There are four K+ channel families:

  1. Voltage-gated K+ (KV)

  2. Calcium-activated (KCa)

  3. Inwardly rectifying K+ (Kir)

  4. Two-pore potassium (K2P) channels.

• These channels share a highly conserved selectivity filter within the pore but have different gating mechanisms.

• They play essential roles in controlling neuronal excitability.

Inwardly Rectifying K+ Channels

• Each subunit consists of two transmembrane domains separated by a pore-forming region.

• These subunits form tetramers (four subunits) to produce functional Kir channels.

• They show strong inward rectification (K+ moves more easily into the cell than out).

• They are typically active around $E_K$, helping to set and maintain the resting membrane potential but close when Vm is depolarized to avoid opposing membrane excitation.

Two-Pore K+ Channels

• These channels have four transmembrane domains and two pore (P) domains per subunit and are referred to as “tandem” or “twin” pore K+ channels (K2P).

• The functional channel is thought to consist of a dimer.

• These channels contribute to “leak” K+ conductances.

• They are regulated by various stimuli, such as pH, O2 partial pressure, membrane stretch, temperature, G-proteins, fatty acid, and inhalation anesthetics.

Calcium-Activated K+ Channels

• These share a similar structure to voltage-gated K+ channels but have an extra transmembrane domain (S0) involved in regulation by β subunits.

• These channels are regulated not only by voltage but also by intracellular $Ca^{2+}$; BKCa channels possess a “calcium bowl” region at the C-terminus, while SK/IKCa channels are modulated by the calcium-binding protein calmodulin.

Voltage-Gated K+ Channels (KV Channels)

• These channels possess six transmembrane domains per subunit with a voltage sensor on the fourth transmembrane segment (S4), allowing them to detect and open in response to membrane depolarization.

• They tend to play roles in repolarizing membranes in nerve and muscle cells, controlling action potential frequency and duration.

• Four α-subunits come together to form the pore region of the channels, and α-subunits usually associate with accessory β subunits, too.

• While all K+ channels differ in gating mechanisms, they share the same tetrameric architecture with a single pore.

Ionic Distribution and Forces at Rest

• There is a high concentration of K+ inside the neuron.

• Both concentration and electrostatic forces push $Na^+$. Thus, $Na^+$ leaks inside across the membrane.

• The $Na^+$ leaking into the neuron is sufficient to bring the Vm away from $E_K$ to the resting state of –65 mV.

Outward K+ Currents

• Anions (A–) are too large to fit through the channels.

• Diffusion pushes K+ down its concentration gradient.

• As K+ leaves, the inside of the cell becomes more negative.

• The difference in electric potential starts pulling K+ back in!

• An equilibrium is reached and no net K+ leaves the cell even though channels are open.

Maintaining Concentration Gradients

• The sodium/potassium pump helps to ensure that concentration gradients are maintained.

• Approximately 70% of all ATP produced by mitochondria is dedicated to keeping the pumps running.

• The pump binds 3 $Na^+$ ions and ATP inside the cell, changing the protein's conformation.

• It releases $Na^+$ outside and picks up 2 $K^+$ ions.

• Binding of $K^+$ to the pump changes its conformation and brings the $K^+$ into the neuron.

K+ Currents in Different KV Channels

• Given the high concentration of potassium inside a neuron and the large ionic driving force, potassium is forced out of a neuron when KV channels are activated.

• Different types of KV channels have different activation and inactivation kinetics.

• In response to depolarizing voltage steps, the current deflections for KV channels are positive, indicating that the current is flowing out of the neuron.

Location of KV Channels in Neurons

• KV1.1 channels in the axon and terminal regulate excitability, action potential propagation, and synaptic transmission.

• KV4.3 channels in dendrites constrain back-propagating action potentials in the dendritic tree.

• KV7.2/7.3 channels dampen excitability and repetitive firing in neurons.

• KCa1.1 channels terminate the action potential and generate after-hyperpolarizations that close $Ca^{2+}$ channels to stop synaptic transmission.

Voltage-Gated Potassium Channels (Part 2)

Review of Voltage-Gated Potassium Channels

• The lecture continues focusing on the structure and function of voltage-gated potassium channels (KV channels).

• Topics include:

  • Classification of different potassium channels, including the role of KV channels in the action potential.

  • KV channel structure, voltage sensitivity, and ion selectivity.

  • Inactivation and phosphorylation of KV channels, including toxins affecting KV channel function.

Structure and Function of KV Channels

• KV channels possess six transmembrane domains per subunit with a voltage sensor on the fourth transmembrane segment (S4), allowing them to detect and open in response to membrane depolarization.

• As such, they tend to play roles in repolarizing membranes in nerve and muscle cells, thus controlling action potential frequency and duration.

• Four α-subunits come together to form the pore-forming region of the channels, and α-subunits usually associate with accessory β subunits to form functional channels.

Alpha Subunits and Pore Domain

• Homotetramer KV channels consist primarily of four identical alpha subunits.

Activation and Permeability

• How are KV channels activated, and why are they permeable only to potassium?

Voltage Sensor Domain (VSD)

• Similar to $Na_V$ channels, conserved arginine residues repeating every 3 amino acid sequences along S4 constitute the main voltage-sensing region in KV channels.

Models of KV Gating

• Sliding Helix Model: Channel gating relies on the charged S4 segments moving, in relation to the channel protein, across the membrane.

• Paddle Model: The gating charge is carried by paddles, composed of an α-helical hairpin formed by S3 and S4, that pivot against the membrane like levers, directly causing channel activation.

• Transporter-Like Model: The charged residues on S4 do not translocate across the membrane but pivot along their longitudinal axis, transporting the gating charge from an extracellular to an intracellularly connected water crevice, and coupling to the opening and closing of the ion channel.

Voltage Sensor Structure

• The structure includes a single monomer depicting the voltage-sensor domain (VSD) and the pore domain (A).

• Arginine residues R1, R2, R3, & R4 are visible on S4 (A).

• Possible trajectories for the gating charges are shown in B.

Pore Domain Structure

• The K+ pathway contains two barriers for K+ that function as a gate:

  1. The selectivity filter (SF) allows the passage of K+ ions, which have shed their hydration shell.

  2. The bundle crossing of the M2/S6 helices (BC gate) forms a barrier for hydrated K+.

Selectivity Filter

• The selectivity filter is highly conserved.

• The sequence alignment of the inner pore helix of the pore module (S6 and M2 segment, respectively) and the P-loop that forms the channel’s selectivity filter contains the TVGYGD signature sequence (highlighted in orange).

• The highly conserved glycine residue in the middle of the inner pore helix is highlighted in red.

Components of the Selectivity Filter

• The main constituents of the filter are oxygen atoms from amino-acid residues Thr–Val–Gly–Tyr–Gly, which are part of the structure of the pore loop.

• The selectivity process is a series of stereochemical checkpoints. Each checkpoint consists of four oxygen atoms that occupy the corners of a square.

• This oxygen-lined checkpoint is repeated five times every ~3.0 Å along the filter.

Ion Selectivity Mechanism

• Potassium ions lose their spheres of hydration to fit through the pore. Sodium ions, in contrast, retain theirs and cannot pass through, due to size and charge considerations.

Inactivation of KV Channels

• Like $Na_V$s, KV channels also inactivate. Potassium flow through open KV channels slows considerably a few msec after activation, despite continued depolarization, indicating that the channels have inactivated.

Voltage-Gated Potassium Channels (Part 3)

Review of Previous Topics

• The lecture continues focusing on the structure and function of voltage-gated potassium channels (KV channels).

• Topics include:

  • Classification of different potassium channels, including the role of KV channels in the action potential.

  • KV channel structure, voltage sensitivity, and ion selectivity.

  • Inactivation and phosphorylation of KV channels, including toxins affecting KV channel function.

KV Channel Inactivation Mechanisms

• Two methods to inactivate KV channels:

  1. N-type inactivation due to the plugging of the pore after opening the cytoplasmic activation gate.

  2. C-type inactivation by collapsing the selectivity filter gate.

N-Type Inactivation

• N-type inactivation (a.k.a., hinged-lid inactivation or ball-and-chain inactivation).

• The inactivation gate is formed by the hydrophobic residues (inactivation ball) on the N-terminus of the alpha subunit, which binds to the central cavity of the pore of KV channels.

C-Type Inactivation

• A slow process, resulting from conformational changes in the selectivity filter in the pore domain, causing it to collapse.

• Collapsing the selectivity filter is a way to stop K+ from leaving.

Modulation of KV Channels

• Auxiliary β subunits regulate KV channels by:

  • Influencing KV channel surface expression

  • Regulating N-type inactivation

  • Playing a role in redox sensing

PIP2 Regulation

• Phosphatidylinositol 4,5-bisphosphate (PIP2) prevents N-type inactivation, regardless of whether the fast inactivation gate was part of the pore-forming alpha subunit or accessory beta subunit.

• PIP2 is permissive in allowing the S4-S5 linker to move freely.

• Depleting PIP2 shifts the voltage-dependence of activation and reduces the open probability of KV channels, resulting in an overall decrease in the amount of current conducted.

Phosphorylation

• Phosphorylation of potassium channels affects their function and plays a major role in regulating cell physiology.

• Dynamic and reversible changes in KV structure and function come about through posttranslational modification via cycles of phosphorylation and dephosphorylation by a wide variety of protein kinases and protein phosphatases.

Toxins Affecting KV Channel Function

• Most toxins from animal venoms block the central pore to prevent K+ transport, while spider toxins regulate the voltage sensors.

• Pore-blocking toxins and chemicals bind to the outer pore of KV channels, including:

  • Dendrotoxin (αDaTX, snake venom)

  • Stichodactyla toxin (ShK, sea anemone)

  • Charybdotoxin (ChTX; 37 AA peptide, scorpion venom)

• Some toxins block deep within the inner pore:

  • Correolide (from extracts of the Costa Rican tree, Spachea correa): Binds to the intracellular cavity of KV1.3 and acts as an immunosuppressant.

  • Tetrabutylammonium (TBA)

  • d-Tubocurarine

• Gating-modifier toxins are hydrophobic and target the voltage sensor:

  • Hanatoxin (35AA): from tarantula, targets KV2.1, KV4.2, producing localized pain, itching

  • Guangxitoxin (GxTx1E): from tarantula Plesiophrictus guangxiensis, targets KV2.1, promoting insulin secretion (potential drug for diabetes).

Potassium Channels in Disease

• K+ channels play a role in many diseases:

  • Epilepsy, memory disorders, chronic pain, cardiac and brain ischemia, hypertension, autoimmune diseases, and cancer.

• Activation of K+ channels reduces excitability; inhibition of K+ channels increases excitability.

• Key roles include regulation of neuronal and cardiac electrical patterns, neurotransmitter release, muscle contractility, hormone secretion, secretion of fluids, and modulation of signal transduction pathways in non-excitable cells.

• Examples of Neuronal K+ Channelopathies include:

  • Episodic ataxia, Spinocerebellar ataxia, Temporal lobe epilepsy, Autism spectrum disorders, Generalized epilepsy, SeSAME syndrome, Acquired neuromyotonia