Ion Channels and Electrophysiology: Voltage Clamp, Patch Clamp, and Action Potentials

How did they use the voltage clamp experiment to determine which ions go which direction?

When they vary external Na+, the initial inward current disappears with decreased Na+ outside, but the outward current remains the same. Pharmacologically blocking Na+ channels with tetrodotoxin eliminates the inward current; blocking K+ channels with tetraethylammonium blocks the potassium current.

In an action potential, Na+ conductance rises and declines _____ (slowly/quickly).

Quickly.

In an action potential, K+ conductance _____ (slowly/quickly) rises and _____ (does not/then) inactivate.

Slowly; does not inactivate.

In an action potential, both Na+ and K+ currents are _____. _____ currents precede _____ currents.

Voltage-dependent; Na+ currents precede K+ currents.

Why does Na+ conductance decline so quickly?

Na+ channels transition through three conformations: closed → open → inactivated.

Name the variations of the patch clamp methods.

Cell-attached recording; whole-cell recording; inside-out recording; outside-out recording.

Cell-attached recording

Allows recording of a single or a few channels.

Whole-cell recording

Suction creates a hole in the plasma membrane, allowing measurement of currents from all channels in the cell.

Inside-out recording

A membrane patch is removed so the cytoplasmic side is exposed; useful for testing cytoplasmic ligands.

Outside-out recording

The membrane patch reseals with the extracellular surface exposed; useful for testing external fluid/extracellular ligands.

Microscopic currents

Current flowing through a single ion channel.

Macroscopic currents

Current flowing through multiple ion channels.

What do microscopic and macroscopic currents of K+ and Na+ channels tell us about channel opening?

K+ and Na+ channel opening is probabilistic and can change as a function of voltage.

What are the two types of active transporters?

ATPase pumps require energy for ion translocation from ATP hydrolysis. Ion exchangers do not use ATP directly; they use an electrochemical gradient as an energy source.

Na+/K+ pump

An ATPase pump. Imports K+ into the cell and exports Na+ out of the cell, maintaining high intracellular K+ and low intracellular Na+.

Ca2+ pump

An ATPase pump that removes Ca2+ from the cytoplasm, including by exporting Ca2+ outside the cell.

Antiporters/exchangers

Move substances in different directions. Examples: Na+/Ca2+ exchanger removes intracellular Ca2+; Na+/H+ exchanger helps control pH.

Co-transporters

Move substances in the same direction. Examples: Na+/K+/Cl− cotransporter moves ions inward; K+/Cl− cotransporter moves ions outward; Na+/neurotransmitter cotransporter moves substances inward.

Translocation of Na+ and K+ by the Na+/K+ ATPase

Stage 1: pump faces inward, releases 2 K+, binds 3 intracellular Na+ and ATP. Stage 2: ATP is hydrolyzed and the pump is phosphorylated and occluded with 3 Na+. Stage 3: pump faces outward, releases 3 Na+ and binds 2 extracellular K+. Stage 4: phosphate is lost; pump becomes occluded with 2 K+ and returns to Stage 1.

Ion channels

Large integral membrane proteins that form an aqueous pore through which ions move across the membrane using their electrochemical gradients. Channels have different mechanisms of gating and selectivity.

Voltage-gated ion channels

Channels that open or close in response to a change in membrane potential.

Ligand-gated ion channel

A channel that opens or closes in response to an extracellular or intracellular ligand.

Mechanically gated channels

Channels that open or close in response to stretching or other mechanical deformation.

Structural similarities and differences between Na+, Ca2+, and K+ channels

Na+, Ca2+, and K+ channels are structurally similar and contain one pore. K+ channels consist of 4 separate subunits; Na+ and Ca2+ channels contain homologous domains within a larger protein. Cl− channels have a different structure and contain two pores.

What are the two main domains of a K+ channel?

Voltage-sensing domain, where voltage changes are sensed; pore domain, where K+ is permitted to pass.

How does a K+ channel ensure ion selectivity?

The pore is sized so very large ions cannot fit, while smaller ions such as Na+ are not properly stabilized. At the narrow selectivity filter, carbonyl oxygen groups stabilize dehydrated K+ and compensate for loss of its hydration shell.

How are ions pushed through the pore domain/selectivity filter?

Electrostatic repulsion between ions.

What does the selectivity filter do?

Ensures that specific ions can pass through the channel; ions must become dehydrated or partially dehydrated to move through the narrow filter.

How does the voltage-sensor domain operate?

The S4 helix contains positively charged residues. Hyperpolarization pulls the voltage sensor inward and favors channel closure; depolarization moves the voltage sensor outward and promotes channel opening.

Functional states of a voltage-gated Na+ channel

Stage 1: closed at resting membrane potential. Stage 2: membrane reaches threshold and Na+ channel opens, allowing Na+ influx. Stage 3: near the peak of the action potential, the Na+ channel inactivates. Stage 4: channel returns to the closed conformation.

Functional states of a voltage-gated K+ channel

Stage 1: closed at resting membrane potential. Stage 2: remains closed early during depolarization. Stage 3: opens near the action potential peak. Stage 4: remains open as the membrane repolarizes/hyperpolarizes. Stage 5: closes as resting membrane potential is restored.

Why do Na+ channels activate and inactivate quickly?

Only 3 of the 4 voltage-sensing domains are required for activation, while K+ channels require all 4. Movement of the slower fourth voltage-sensing domain is associated with fast Na+ channel inactivation.

Kv2.1

Regular voltage-gated potassium channel.

Kv4.1

Voltage-gated potassium channel with an inactivation phase.

HERG

A K+ channel found in heart cells that opens relatively late and closes quickly.

Inward rectifier K+ channels

Open more readily when membrane voltage is negative and close more at positive voltages; these channels contribute to maintaining membrane voltage.

How can optogenetics be used to manipulate neural activity?

Deliver genes into neurons that express light-activated proteins. Shining light can alter ion flow and neuronal firing, providing temporal and spatial control of neural activity. It can be used bidirectionally on single neurons, local circuits, or distributed neural networks.

What properties did Hodgkin and Huxley propose ion channels must have?

Ion channels must allow ions to flow at high rates, use electrochemical gradients, be selective for specific ions, and detect/respond to changes in membrane voltage.

What provided the first evidence for voltage-sensitive, ion-selective channels?

The patch clamp method developed by Erwin Neher and Bert Sakmann.

Voltage clamp vs. patch clamp

Voltage clamp measures aggregate/macroscopic current flowing through thousands of channels, while patch clamp can measure current through individual ion channels.

Approximately how large is the current through a single ion channel?

About 1-2 pA; this is called a microscopic current.

How did researchers isolate currents from single Na+ channels during patch clamp experiments?

They performed patch clamp recordings in the presence of K+ channel blockers.

What happens to microscopic Na+ current at membrane potentials below and above ENa?

At membrane potentials more negative than ENa, Na+ current is inward. At membrane potentials more positive than ENa, the current reverses and becomes outward.

What happens to microscopic Na+ current when extracellular Na+ concentration is reduced?

The size of the microscopic Na+ current decreases.

How did single-channel recordings support the idea that microscopic Na+ currents produce macroscopic Na+ current?

Individual Na+ channels open and close probabilistically, but averaging many microscopic recordings produces a trace similar to the macroscopic Na+ current.

What toxin blocks microscopic Na+ currents?

Tetrodotoxin (TTX), a Na+ channel inhibitor.

What are characteristics of microscopic K+ currents?

They are directed outward when the membrane is depolarized, do not show the rapid inactivation seen in Na+ channels, and can be blocked by K+ channel inhibitors.

Approximately how many genes encode ion channels?

About 200 genes.

What are the four major types of ion channel gating in this module?

Voltage-gated, ligand-gated, temperature-gated, and mechanically gated.

About how many genes encode voltage-gated ion channels?

About 100 genes.

SCN genes

Genes encoding voltage-gated Na+ channels. Humans have about 10 Na+ channel genes, and different channels can have different properties.

CACNA genes

Genes encoding voltage-gated Ca2+ channels. Humans have about 10; Ca2+ can affect membrane potential and activate intracellular signaling pathways.

KCN genes

Genes encoding K+ channels. Humans have about 78, making K+ channels the largest and most diverse ion channel family.

How diverse are K+ channels?

Some K+ channels inactivate, some are sensitive to pH, and some are activated by Ca2+.

CLCN genes

Genes encoding several types of Cl− channels that influence resting membrane potential and neuronal excitability.

What activates an extracellular ligand-gated ion channel?

Binding of an extracellular chemical such as a neurotransmitter to the channel.

What neurotransmitters are examples of extracellular ligands for ligand-gated channels?

Glutamate, dopamine, and acetylcholine.

Can ligand-gated channels be permeable to more than one ion?

Yes. Some ligand-gated channels allow several different ions to pass.

Which ions can pass through an NMDA-type glutamate receptor?

Na+, K+, and Ca2+.

What intracellular molecules can gate ion channels?

Second messengers and intracellular signals such as cAMP, cGMP, Ca2+, and protons.

What is the TRP channel family?

A family of temperature-sensitive ion channels involved in thermosensation; mammals have about 28 TRP genes.

What kinds of temperatures can different TRP channels detect?

Different TRP channels can respond to cold, cool, warm, or hot temperatures.

Examples of temperature-sensitive TRP channels

TRPA1, TRPM8, TRPV3, TRPV4, TRPV1, and TRPV2.

What do mechanically gated ion channels detect?

Mechanical displacement or deformation such as stretch, touch, pressure, and forces involved in hearing.

Examples of mechanically gated channel families

Piezo, TRP, TMEM, and TREK channels.

What is the basic structure of a bacterial K+ channel?

Four subunits assemble to form a central pore that is selectively permeable to K+.

How does the structure of a Na+ channel compare with a K+ channel?

Na+ channels are larger and contain 24 transmembrane alpha helices, but their overall organization resembles the four-part structure of K+ channels.

How are Cl− channels structurally different from Na+ and K+ channels?

Cl− channels contain two monomers, and each monomer contains its own Cl−-permeable pore.

Why can't an ion larger than K+ pass through a K+ selectivity filter?

It is physically too large to fit through the pore.

Why can't Na+ pass easily through a K+ selectivity filter even though Na+ is smaller than K+?

The filter is sized to stabilize dehydrated K+. Its walls are too far apart to properly stabilize the smaller dehydrated Na+ ion.

What is the role of the water-filled cavity in a K+ channel?

It helps trap hydrated K+ ions before they enter the selectivity filter.

What stabilizes dehydrated K+ inside the K+ selectivity filter?

Electronegative oxygen atoms in the selectivity filter.

Why can several K+ ions move rapidly through a selectivity filter?

Multiple K+ ions can occupy the filter at once, and electrostatic repulsion between their positive charges helps push the ions through.

How does Na+ pass through a Na+ channel pore?

The Na+ channel has a wider pore than a K+ channel and allows partially dehydrated Na+ ions to enter; multiple ions in the pore help push one another through.

How does a Cl− channel attract Cl− into its pore?

The pore is lined with positive charges that attract negatively charged Cl− ions.

What is the difference between ion channel permeability and gating?

Permeability determines which ions can pass through the channel; gating determines when the channel opens or closes.

What is the S4 helix in a voltage-gated channel?

A transmembrane region containing positively charged residues that acts as a voltage sensor.

How does depolarization affect the voltage sensor of a voltage-gated channel?

Depolarization moves the positively charged voltage sensor outward, which promotes opening of the channel pore.

How does hyperpolarization affect the voltage sensor?

Hyperpolarization moves the voltage sensor inward, which promotes channel closure.

What ligand-gated channel is used as the main example of extracellular chemical gating?

The AMPA glutamate receptor.

What is the structure of an AMPA receptor?

It is made of four subunits whose transmembrane regions form the channel pore and gate.

What happens to an AMPA receptor when no glutamate is present?

The channel gate blocks the pore and the channel remains closed.

How does glutamate open an AMPA receptor?

Glutamate binds to an extracellular clam shell-shaped ligand-binding domain, causing the clam shell to close and mechanically open the channel gate.

Is glutamate an extracellular or intracellular ligand for AMPA receptors?

Extracellular.

What is an example of a channel gated by intracellular ligands?

Cyclic nucleotide-gated channels activated by cAMP or cGMP.

Are cAMP and cGMP extracellular or intracellular signals?

Intracellular second messengers.

How do cAMP and cGMP open cyclic nucleotide-gated channels?

They bind to large cytoplasmic ligand-binding domains, producing a conformational change in a linker that opens the pore.

What is the basic structure of cyclic nucleotide-gated channels?

Four subunits assemble to form a pore, similar to voltage-gated K+ channels.

What type of pore do TRP channels form?

TRP channels generally consist of four subunits that form a cation-selective pore.

How can heat or capsaicin open a TRP channel?

Heat or capsaicin can displace phosphatidylinositol membrane lipids near a helical linker, causing the linker to move and the channel to open.

Capsaicin activates what type of channel mechanism?

Temperature-sensitive TRP channel gating.

What is Piezo?

A mechanically gated ion channel important for mechanosensation.

What is unusual about the structure of Piezo channels?

They are very large channels with about 38 transmembrane domains, located in curved regions of the membrane.

What are the main structural parts of a Piezo channel?

A central cation-permeable pore and three blade-like sensor regions that detect membrane curvature.

How does mechanical force open a Piezo channel?

Mechanical force flattens the curved membrane, moving the Piezo blades/sensors and causing the central pore to open.

Channelrhodopsin-2 (ChR2)

A light-sensitive channel used in optogenetics to generate electrical activity in neurons.

Halorhodopsin (NpHR)

A light-sensitive protein used in optogenetics to inhibit electrical activity in neurons.

What light activates ChR2 in the Module 2 figure?

Blue light.

What light activates NpHR in the Module 2 figure?

Yellow light.

What is the main purpose of optogenetics?

To use light-sensitive proteins to control neuronal electrical activity with precise spatial and temporal control.