L2a - Electrophysiology (1) Flashcards
Define ‘equilibrium potential’.
The potential gradient across the membrane needed to maintain concentration gradient / needed to stop diffusion down chemical gradients.
What is the membrane potential of a resting neurons primarily determined by?
Movement of K+ ions across the membrane.
Describe the state of potassium ions in the cell at starting / resting state.
- Interior of the cell has a much higher concentration of potassium than the exterior
- K+ ions diffuse out, down the concentration gradient
- As K+ leave the cell, the inside becomes more negative
For any given ion, x, what is the total chemical potential?
RTln[x] + zx FV
What is the total chemical potential of a given ion governed by?
- Ion concentration
- Valency
Describe what happens to chemical potential at equilibrium.
intracellular chemical potential = extracellular chemical potential
What does the influence of ionic gradients on membrane potential depend on?
Relative permeability of the membrane to each ion
Describe the membrane potential of most cells at rest.
Most cells (at rest) will have a membrane potential closer to that of potassium, because the rest of the cell is most permeable to potassium ions - and impermeable to sodium and calcium ions.
State which equipment is needed to record a biphasic response from a squid axon.
- Earth (ground) electrode
- Intracellular (recording) electrode
- External solution
What happens when the external sodium is lowered?
Peak of action potential is lowered
the electrochemical driving force is lower for sodium ions
State which equipment is needed for a voltage clamp recording from a squid axon (an action potential).
- Earth (ground) electrode
- Intracellular (V clamp) electrode
- Intracellular (recording) electrode
- External solution
What is done to prepare the electrodes for a voltage clamp?
Electrodes are varnished to prevent contact with each other and the external solution
What does a voltage clamp allow?
Allows internal voltage of cell / axon to be set at a fixed value, so electrical current flowing in / out of neurons can be measured
What needs to happen in order for neurones to generate electrical impulses?
They need to be at a voltage different that the environment outside the neurone
Which voltage gated channels activate more slowly and do not show inactivation in axons?
Voltage-gated potassium ion channels (K+)
Describe the trace showing the potential inside the membrane.
A) -65 mV resting potential
B) > 0 mV step (voltage clamp)
C) -65 mV resting potential
Describe the trace showing the measured transmembrane current.
1) Capacitive current
2) Transient inward current (Na+)
3) Delayed outward current (K+)
What happens when the intracellular potassium is lowered?
Peak potassium current is decreased
In a clamped membrane potential trace, there is a sodium current and a potassium current. What differs between both?
They activate at different times
Why does capacitive current occur?
The step from one potential to another alters the charge separation and thus, the electrical potential difference across the membrane
What happens when a new potential is reached?
No more capacitive current
What is the magnitude of current dictated by?
- Number of channels that will pass current (how many are present and open)
- The electrochemical gradient (difference between the reversal and membrane potentials)
What do positive and negative ions leaving a cell cause?
- Positive ions leaving a cell cause a positive current
- Negative ions leaving a cell cause a negative current
What is channel conductance in voltage-gated ion channels determined by?
Changes in membrane potentials
State examples of voltage-gated ion channels.
- Cardiac sodium channels
- L-type calcium channels
What do ligand-gated ion channels open (or close) in response to?
The presence of certain ligands
State examples of ligand-gated ion channels.
- Nicotinic ACh receptors
- ATP gated P2X receptors
Describe 6 other types of gating (excluding voltage-gated and ligand-gated ion channels).
- Inward rectifiers (pass inward current more easily)
- Twin pore (‘leak’) potassium channels
- Mechanosensitive
- Temperature sensitive
- Light sensitive
- Indirectly modulated
What are potassium inward rectifiers blocked by?
Magnesium and spermine at more positive potentials
State examples of mechanosensitive channels.
- Stretch activated chloride current
State examples of indirectly modulated channels.
- G-protein-regulated inwardly rectifying K+ channels
Describe the characteristics of voltage-gated potassium ion channels.
- Requires 4 alpha subunits to form a channel (tetramer)
- Often has associated regulated beta subunits
- Each subunit has 6 transmembrane domains, (TMDs S1-6)
- Pore forming region is between S5 and S6
Describe the characteristics of voltage-gated sodium and calcium ion channels.
Similar in structure to potassium channels:
- 4 alpha subunits needed to form a channel
- Pore forming region is between S5 and S6
- Ion channel is a tetramer of 4 alpha subunits
- Often has associated regulated beta subunits too
- Except 4 homologous domains form a single protein
Describe the S4 transmembrane domain.
- In potassium, sodium and calcium channels: the S4 helices (TMD) is the voltage sensor (causes a channel to activate / deactivate)
- Contain a positively charged amino acid every 3rd residue; typically arginine and lysine
Describe what happens to channels before and after depolarisation, and by what mechanism.
1) Closed before depolarisation
2) Activated after depolarisation
3) And then open after activation
(Mechanical Lever Model)
Describe the steps leading to N-type inactivation.
1) Deactivated: Activation gate closed, non-conducting
2) Activated: Channel undergoes conformational change, both gates open
3) Inactivated: Refractory to open, non-conducting
What happens during inactivation?
- Intracellular residues (inactivation gate) occlude the inside face of the channel at the n-terminus
- Blocks further conduction of ions
- After recovery, channel returns to resting state
What happens during activation and inactivation in terms of membrane potential and relative conductance?
Activation
- Increase in relative conductance
- Increase in membrane potential
Inactivation
- Decrease in relative conductance
- Increase in membrane potential
Define ‘patch clamp’.
Laboratory technique in electrophysiology, used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membranes
How is the sample prepared in the patch clamp method?
1) Glass pipette with small opening is used to make tight contact with tiny area of neuronal membrane
2) After applying a small amount of suction; seal between pipette and membrane is so tight that cytoplasm becomes continuous with the pipette interior
3) All ions passing through the membrane must therefore flow into the pipette
What is the role of the operational amplifier in the patch clamp method?
Record of the current flowing - shows whether the ion channel is open or closed
What is the whole-cell (perforated) patch method?
Pore forming antibiotic in patch pipette perforated the sealed patch of membrane in contact with the pipette
(provides low-resistance electrical access, allowing control of transmembrane voltage)
Describe a current clamp.
- Clamp the current and record voltage (membrane potential)
- Used to record action potentials
Describe a voltage clamp.
- Clamp the voltage
- Record current
Describe the characteristics of a channel that has faster activation / deactivation than inactivation gating.
- At voltage A, the channel is closed (deactivated)
- At voltage B, the channel is open (activated) and then inactivates
What happens when current is negative vs. when current is positive?
Current is negative:
- Influx of positive ions
- Efflux of negative ions
- Moves membrane potential to more positive values
Current is positive:
- Influx of negative ions
- Efflux of positive ions
- Moves membrane potential to more negative values
Describe the graph showing the arrival of an action potential.
1) At a negative membrane potential, Na+ channels open
2) Sodium influx causes an increase in membrane potential to more positive values
3) Na+ channels inactivate
4) An action potential arrives
5) K+ channels open
3) Potassium influx causes a decrease in membrane potential to more negative values
4) Membrane enters refractory period
Describe the characteristics of ligand-gated ion channels.
- Open in response to the binding of a chemical messenger
- Known as ionotropic receptors
What can ligand-gated ion channels be broadly classified as?
- Cys-loop receptors
- Ionotropic glutamate receptors
- ATP-gated channels
Describe the characteristics of cys-loop receptors.
- Named after a loop formed by a disulphide bond between 2 cysteine residues in the N terminal extracellular domain
- Can be either cationic or anionic channels
State the cys-loop receptors that are cationic channels.
- Serotonin (5-HT) Receptors
- Nicotinic Acetylcholine Receptors
- Zinc Activated Receptors
State the cys-loop receptors that are anionic channels.
- GABAa Receptors
- Glycine Receptors (GlyR)
Describe the structure of cys-loop receptors.
- Pentamers of 5 protein subunits
Nicotinic receptor considered to be the ‘typical structure’ but there are variations
What are Nicotinic Acetylcholine Receptors?
Ligand-gated ion channels that bind acetylcholine / nicotine
What are Ionotropic Glutamate Receptors?
- Cation channels
- That bind the neurotransmitter: glutamate
What can Ionotropic Glutamate Receptors be classified as?
- AMPA (GluA)
- Kainate (GluK)
- NMDA (GluN)
- ‘Orphan’ (GluD)
Describe the structure of Ionotropic Glutamate Receptors.
- Composed of 4 large subunits
- Each have 4 discrete domains:
• Amino-terminal domain (ATD)
• Extracellular ligand-binding domain (LBD)
• Transmembrane domain (TMD)
• Intracellular carboxyl-terminal domain (CTD)
Describe the characteristics of ATP-Gated Channels (P2X Receptors).
- Activated by 3x ATP molecules
- 7 different subtypes (P2X 1-7)
- Cation channels (typically monovalent cations, but some will carry calcium ions)
What variety of tissues can ATP-Gated Channels (P2X Receptors) hé expressed in?
- Vas deferens
- Platelets
- Neurons
- Leukocytes
- Smooth muscle
How do you obtain a trace of the current passing through ATP-Gated Channels (P2X Receptors)?
- Voltage clamp
1) Hold at set voltage
2) Add ligand
Describe what happens at a neuromuscular junction.
1) Action potential arrives at axon terminal of motor neurone
2) Voltage-gated Ca2+ channels open. Ca2+ enters the axon terminal moving down its electrochemical gradient.
3) Ca2+ entry causes ACh (neurotransmitter) to be released by exocytosis
4) ACh diffuses across the synaptic cleft and binds to its receptors on the sarcolemma.
5) ACh binding opens ion channels in the receptors that allow the simultaneous passage of Na+ into the muscle fiber. More Na+ ions enter than K+ ions exit, which produces a local change in the membrane potential called the end plate potential.
6) ACh effects are terminated by its breakdown in the synaptic cleft by acetylcholinesterase and diffusion away from the junction.
