Comprehensive Notes on Ion Channels and Transporters

Ion Channels and Transporters

The Patch Clamp Method

• Cell-Attached:
  • Pipette seals to the membrane without breaking it.
  • Records single-channel activity in an intact cell.
• Whole-Cell:
  • Pipette ruptures the membrane.
  • Records current through channels across the entire cell.
• Inside-Out:
  • Patch is pulled away, exposing the cytoplasmic face.
  • Allows for single-channel recording.
• Outside-Out:
  • Patch is pulled after whole-cell configuration, exposing the extracellular face.
  • Enables single-channel recording.

Patch Clamp Measurements of Single Na+ Channels

• Single-Channel Currents:
  • Depolarization triggers brief Na⁺ currents.
• Inactivation:
  • Channels open in the first 1–2 ms and then inactivate.
• Stochastic Behavior:
  • Individual channel behavior varies.
• Microscopic vs. Macroscopic Currents:
  • Single-channel (microscopic) and whole-cell (macroscopic) currents display similar time courses.
  • Macroscopic currents resemble the average of many microscopic currents.
• Voltage Dependence:
  • Higher depolarization increases the probability of Na⁺ channel opening.

Voltage-Gated Na+ Channel Structure

• Key Components:
  • Voltage sensor
  • Na+ selectivity filter
  • Inactivation gate
  • Activation gate
• Functional States:
  • Closed (Not Activated): Channel is closed at resting membrane potential.
  • Open (Activated): Voltage sensor detects depolarization, opening the activation gate and allowing Na+ ions to flow through the channel, following their electrochemical gradient.
  • Closed (Inactivated): The inactivation gate blocks the channel after a short period of activation.

Patch Clamp Measurements of Single K+ Channels

• Single-Channel Currents:
  • Depolarization triggers brief K⁺ currents.
• Delayed Activation:
  • Channels open after a delay but remain open during depolarization.
• Stochastic Behaviour:
  • Individual channel behavior varies.
• Microscopic vs. Macroscopic Currents:
  • Single-channel and whole-cell currents show similar time courses.
  • Macroscopic currents look like the average of many microscopic currents.
• Voltage Dependence:
  • Higher depolarization increases K⁺ channel opening probability.

Functional States of Voltage-Gated Na+ and K+ Channels

• Resting (Hyperpolarized):
  • Both Na⁺ and K⁺ channels are closed.
• Activation (Depolarized):
  • Na⁺ channels open first, followed by K⁺ channels.
• Inactivation:
  • Na⁺ channels inactivate with prolonged depolarization.
  • Many K⁺ channels remain open.

Detour: Ribbon Diagrams

• Definition: 3D schematic representations of protein structure.
• Purpose:
  • Depict the general course and organization of the protein backbone in 3D.
  • Serve as a visual framework for details of the atomic structure.
• Components:
  • α-helices: coiled ribbons or thick tubes.
  • β-sheets: arrows.
  • Non-repetitive coils or loops: lines or thin tubes.
• Direction:
  • Indicated locally by arrows.
  • May be indicated overall by a colour ramp along the ribbon.

Detour: Peptide Synthesis

• Solid-Phase Peptide Synthesis:
  • A method for chemically synthesizing peptides.
  • Involves attaching the first amino acid to a resin bead.
  • Fmoc protecting groups are used.
  • Deprotection and coupling steps are repeated to add subsequent amino acids.
  • The final step involves uncoupling the peptide from the linker using TFA (trifluoroacetic acid).

Detour: Protein Folding

• Definition: The physical process by which a protein changes from a linear chain of amino acids to a three-dimensional structure.
• Importance: Essential for biological function or activity.
• Process:
  • Folding often begins during translation.
  • Amino acids interact to produce a native state.
• Native State:
  • Determined by the amino-acid sequence (primary structure).
• Misfolding:
  • Can lead to inactive proteins or proteins with modified/toxic functionality.
  • Associated with neurodegenerative diseases due to the accumulation of amyloid fibrils.
  • Can cause allergies due to incorrect protein structures.
• Denaturation:
  • Transition from a folded to an unfolded state.
  • Occurs in cooking, burns, and proteinopathies.
• Folding Time:
  • Varies dramatically depending on the protein.
  • Small proteins can fold in milliseconds or microseconds.
• Computational Studies:
  • Understanding and simulating protein folding has been a challenge since the 1960s.

Structure of a Simple Bacterial K+ Channel

• Subunit Structure:
  • Two membrane-spanning domains and a pore loop.
• Channel Architecture:
  • Four subunits form the channel.
  • Top view shows K⁺ ion in the pore.
• Permeation Pathway:
  • Large aqueous cavity leads to a narrow selectivity filter.
• Ion Selectivity:
  • Negative charges guide K⁺ ions through the filter after dehydration.

Structure of a Mammalian Voltage-Gated K+ Channel

• Subunit Structure:
  • Four subunits, each with a transmembrane and T1 domain.
  • β subunits attached.
• Channel Architecture:
  • Separate voltage-sensing and K⁺-conducting pore domains.
• Voltage-Dependent Gating:
  • Depolarization moves the voltage sensor up, pulling the linker to open the pore.
  • Hyperpolarization moves the sensor down, closing the pore.
• Voltage Sensor Movement:
  • Paddle-like sensor shifts outward with depolarization and inward with hyperpolarization.

Types of Voltage-Gated Ion Channels

• Types:
  • Na⁺, Ca²⁺, K⁺, Cl⁻ Channels (selectively permeable to specific ions).
• Structure:
  • Multiple transmembrane domains form pores.
• Selectivity Filter:
  • Determines which ions pass through.
• Voltage Sensing & Gating:
  • Controls channel opening/closing based on membrane potential.
• Channel Similarities:
  • Na⁺, Ca²⁺, and K⁺ channels share structural features.

Diverse Properties of K+ Channels

• Kᵥ2.1:
  • Little inactivation.
  • Involved in action potential repolarization.
• Kᵥ4.1:
  • Inactivates during depolarization.
  • Regulates firing intervals.
• HERG:
  • Inactivates rapidly.
  • Current flows at the end of depolarization.
• Inward Rectifiers:
  • Preferentially conduct K⁺ at hyperpolarized potentials.
• 2-P K⁺ Channels:
  • Respond to chemical signals (e.g., pH), not voltage.
• Ca²⁺-Activated K⁺ Channels:
  • Open in response to intracellular Ca²⁺ and sometimes depolarization.

Ligand-Gated Ion Channels

• Neurotransmitter-Gated:
  • Activated by extracellular glutamate.
• Proton-Gated:
  • Activated by extracellular H⁺.
• Second Messenger-Gated:
  • Activated by intracellular Ca²⁺.
• Cyclic Nucleotide-Gated:
  • Activated by cAMP or cGMP.

Thermosensitive and Mechanosensitive Channels

• Thermosensitive Channels:
  • Open in response to heat.
  • Allow ion flux through an open pore.
• Mechanosensitive Channels:
  • Respond to mechanical force.
  • Blades or other structures open the pore to allow ion flux.

Active Transport Pumps

• Ion Concentrations:
  • Inside: $Na^+$ (5-15 mM), $K^+$ (140 mM), $Cl^-$ (4 mM), $Ca^{2+}$ (0.1 μM)
  • Outside: $Na^+$ (145 mM), $K^+$ (5 mM), $Cl^-$ (110 mM), $Ca^{2+}$ (2.5-5 mM)
  • A⁻ (impermeant anions): Inside (147 mM), Outside (25 mM)
• Equilibrium Potentials (Nernst Potentials):
  • $Na^+$: $E_{Na} = 62 \log \frac{145}{15} = 90 \text{ mV}$
  • Simplified Calculation: $E_{Na} = 61 \text{ mV}$
  • $K^+$: $E_K = 62 \log \frac{5}{140} = -90 \text{ mV}$
  • $Cl^-$: $E_{Cl} = -62 \log \frac{110}{4} = -89 \text{ mV}$
  • $Ca^{2+}$: $E_{Ca} = 31 \log \frac{2.5 \times 10^{-3}}{10^{-4}} = 136 \text{ mV}$
  • Simplified Calculation: $E_{Ca} = 31 \log \frac{5}{10^{-4}} = 146 \text{ mV}$
    *Note: Temperature T = 37°C (310°K)

Examples of ATPase Pumps

• (A) Na+/K+ Pump:
  • Uses ATP to transport $Na^+$ out and $K^+$ in.
  • Involves phosphorylation and conformational changes.
• (B) Ca2+ Pump:
  • Pumps $Ca^{2+}$ into the lumen of the sarcoplasmic reticulum.
  • Also uses ATP hydrolysis.

Ion Movements Due to the Na+ Pump

• Normal Pump Action:
  • $Na^+$ is pumped out and $K^+$ is pumped in.
• Experimental Manipulations:
  • Removing $K^+$ breaks the pump.
  • Replacing $K^+$ fixes the pump.
  • Blocking ATP breaks the pump.
  • Recovering ATP fixes the pump.

Translocation of Na+ and K+ by the Na+ Pump

• (A) Steps:
  1. Conformation change allows $K^+$ release and $Na^+$ binding.
  2. $Na^+$ bound but occluded.
  3. Conformation change causes $Na^+$ release and $K^+$ binding.
  4. $K^+$ bound but occluded.
• (B) States:
  • $Na^+$ bound state.
  • $K^+$ bound state.

Examples of Ion Exchangers

• Energy Source:
  • Use electrochemical gradients of co-transported ions.
• Antiporters:
  • Swap ions across the membrane.
• Co-Transporters:
  • Move multiple ions in the same direction.