Ion Channels and Transporters Notes

Ion Channels and Transporters

Patch Clamp Method

The patch clamp method is a technique used to study ion channel activity. There are several configurations:

Cell-Attached: The pipette seals to the cell membrane without breaking it, allowing recording of single-channel activity in an intact cell.
Whole-Cell: The pipette ruptures the cell membrane, enabling recording of current through channels across the entire cell.
Inside-Out: A patch of membrane is pulled away from the cell, exposing the cytoplasmic face. This allows for single-channel recording.
Outside-Out: A patch of membrane is pulled away after establishing whole-cell configuration, exposing the extracellular face. This also allows for single-channel recording.

Patch Clamp Measurements of Single Na+ Channels

Patch clamp experiments on squid giant axons reveal insights into single Na+ channel behavior:

Single-Channel Currents: Depolarization triggers brief inward Na+ currents.
Inactivation: Na+ channels open quickly (1-2 ms) and then inactivate.
Stochastic Behavior: Individual channels exhibit variability in their behavior.
Microscopic vs. Macroscopic Currents: Macroscopic currents, recorded across the entire cell, resemble the average of many single-channel (microscopic) currents. They display similar time courses.
Voltage Dependence: Increased depolarization raises the probability of Na+ channel opening.

Voltage-Gated Na+ Channel Structure

The voltage-gated Na+ channel possesses several key structural features:

Voltage Sensor: Detects changes in membrane potential.
Na+ Selectivity Filter: Permits passage of Na+ ions.
Inactivation Gate: Blocks the channel.
Activation Gate: Opens the channel upon depolarization.

During depolarization, the voltage sensor triggers the opening of the activation gate, allowing Na+ ions to flow down their electrochemical gradient. Subsequently, the inactivation gate closes, halting the flow.

Patch Clamp Measurements of Single K+ Channels

Similar experiments on K+ channels reveal distinct properties:

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

Functional States of Voltage-Gated Channels

Voltage-gated Na+ and K+ channels cycle through different functional states depending on the membrane potential:

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

Ribbon Diagrams

Ribbon diagrams are 3D schematic representations of protein structure and are one of the most common methods of protein depiction used today. Alpha-helices are represented as coiled ribbons or thick tubes, beta-sheets as arrows, and non-repetitive coils or loops as lines or thin tubes.

Protein Folding

Protein folding is the physical process by which a protein, after synthesis by a ribosome as a linear chain of amino acids, changes from an unstable random coil into a more ordered three-dimensional structure. This structure permits the protein to become biologically functional or active.

The correct three-dimensional structure is essential to function, although some parts of functional proteins may remain unfolded, indicating that protein dynamics are important.

Structure of a Bacterial K+ Channel

The structure of a bacterial K+ channel, determined by crystallography, reveals:

Subunit Structure: Two membrane-spanning domains and a pore loop.
Channel Architecture: Four subunits form the channel, with a K+ ion in the pore.
Permeation Pathway: A 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 voltage sensor up, pulling linker to open pore; hyperpolarization moves sensor down, closing pore.
Voltage Sensor Movement: Paddle-like sensor shifts outward with depolarization, inward with hyperpolarization.

Types of Voltage-Gated Ion Channels

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 depolarization end.
Inward Rectifiers: Preferentially conduct K⁺ at hyperpolarized potentials.
2-P K⁺ Channels: Respond to chemical signals (e.g., pH), not voltage.
Ca²⁺-Activated K⁺: Open in response to intracellular Ca²⁺ and sometimes depolarization.

Ligand-Gated Ion Channels

Neurotransmitter-Gated: Activated by extracellular neurotransmitters like 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

These channels respond to temperature and mechanical stimuli, respectively.

Ion Concentrations and Equilibrium Potentials

The table below shows the intracellular and extracellular concentrations of key ions in a typical mammalian neuron, along with their Nernst equilibrium potentials at 37°C (310°K).

Ion	Intracellular (mM)	Extracellular (mM)	Nernst Potential (mV)	Calculation
Na+	5-15	145	$+61$	$62 \log_{10} \frac{145}{15}$
K+	140	5	$-90$	$62 \log_{10} \frac{5}{150}$
Cl-	4	110	$-89$	$-62 \log_{10} \frac{110}{4}$
Ca2+	0.1 μM	2.5-5	$+136 to +146$	$31 \log<em>{10} \frac{2.5}{10^{-4}}$ to $31 \log</em>{10} \frac{5}{10^{-4}}$
A-	147	25	N/A	Membrane-impermeant anions

Active Transport Pumps

Na+/K+ Pump: Transports 3 Na+ ions out and 2 K+ ions in, maintaining electrochemical gradients. It is an ATPase pump.
Ca2+ Pump: Removes Ca2+ from the cytoplasm.

Ion Movements due to the Na+ Pump

The activity of the Na+ pump can be manipulated experimentally:

Normal pump action.
Removing extracellular K+ can break the pump.
Replacing K+ can fix the pump.
Blocking ATP can break the pump.
Recovering ATP can fix the pump.

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

The process involves conformational changes and binding/release of ions:

Conformation change allows K+ release and Na+ binding, driven by phosphorylation.
Na+ bound but occluded.
Conformation change causes Na+ release and K+ binding, driven by dephosphorylation.
K+ bound but occluded.

Examples of Ion Exchangers

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