Membranes and receptors 5 Flashcards
What ion channels are present in nerve terminals?
Voltage-gated Na+ channels
Voltage-gated K+ channels
Voltage-gated Ca2+ channels
What effect does an action potential arriving at a nerve terminal have?
Depolarisation opens voltage-gated Ca2+ channels. This is therefore a means by which you can regulated neurotransmitter release from the axon terminal.
Why does the small influx of Ca2+ into the nerve terminal have such a big effect?
Because the intracellular [Ca2+] is kept so low, this influx raises the intracellular concentration significantly.
Voltage-gated Ca2+ channels are structurally similar to what other type of voltage-gated channel?
Na+
Where are phosphorylation sites found on Ca2+ and Na+ voltage-gated channels?
Cytoplasmic side - therefore can be phosphorylated by intracellular messengers e.g. by PKA in sympathetic activation (e.g. to increase heart rate).
Where are glycosylation sites found on Ca2+ and Na+ voltage-gated channels?
Extra-cellularly
In Na+ and Ca2+ voltage-gated channels, which subunit is the pore-forming subunit and what is the function of the other subunits?
The alpha-subunit forms the pore and the other subunits fine-tune the properties and enable correct regulation of channel activity.
What is the neuromuscular junction?
The synapse between a nerve and a skeletal muscular fibre.
Describe the mechanism of neurotransmitter release into the neuromuscular junction.
- Ca2+ entry through Ca2+ channels
- Ca2+ binds to synaptotagmin
- Vesicle brought close to membrane
- Snare complex makes fusion pore
- Transmitter released through this pore
Where are L-type Ca2+ channels found?
Muscles, neurones and lungs
What drug can block L-type Ca2+ channels?
Dihydropyridines e.g. Nifedipine
What is a muscle spindle?
A sensory receptors in the belly of a muscle that primarily detects changes in the length of this muscle and conveys the information via sensory neurones to the CNS. The brain processes this information to determine the position of the body parts.
What type of ion channel is nicotinic acetylcholine receptor channels?
Ligand-gated ion channels (intrinsic ion channels).
Binding of acetyl choline to nicotinic acetyl choline receptor channels causes what to occur?
- Binding of 2 ACh to receptors opens intrinsic channel
- Channel is non-specific to ions and lets Na+ and K+ through (and a small amount of Ca2+)
- Influx of Na+ exceeds efflux of K+, as Na+ is further away from its equilibrium potential
- Depolarisation of the sarcolemma occurs
- This will try to reach reversal potential (no net change of Na+ across the membrane)
What is meant by the reversal potential or Nernst potential?
The membrane potential of an ion at which there is no net (overall) flow of that particular ion from one side of the membrane to the other.
How many ACh need to bind to nicotinic acetylcholine receptor channels to open the ion channel?
Two
What happens to ACh in the synaptic cleft
It diffuses across the cleft can bind to acetylcholine receptor channels but is quickly broken down by acetlycholinesterase.
How does ACh causes nicotinic ACh channels to open?
ACh binds to each alpha-subunits (x2) and causes a conformational change in the receptor which causes the pore to open.
What is the end plate potential?
The depolarizations of skeletal muscle fibers caused by neurotransmitters binding to the postsynaptic membrane in the neuromuscular junction.
What happens to the end plate potential when the external Ca2+ is lowered?
End plate potentials decrease in amplitude, as the equilibrium potential of Ca2+ decreases.
Transmitter release from the pre-synaptic membrane is dependent on what?
Ca2+ entry
How does curare/ d-tubocurarine (a poison used to hunt for food in the amazon) cause paralysis?
It is a compeitive antagonist. It blocks transmission between nerve and muscle fibre by competitively blocking nicotinic acetlycholine receptors. It binds to the receptors but does not open the channel.
How can paralysis by d-tubocurarine (d-TC) be overcome?
It does not bind the acetlycholine receptors forever, therefore high concentrations of acetycholine can overcome this block.
How is a muscle AP triggered from the binding of ACh to nicotinic ACh receptor channels?
- Channels open
- Na+ influx (greater than K+ efflux) depolarises sarcolemma -> end plate potential
- Depolarisation activates adjacent Na+ channels due to local spread of charge and causes the muscle AP.
What is the mechanism of action of depolarising blockers like succinylcholine?
They bind to ACh receptors and cause the channels to open and remain open. This continued depolarisation leads to inactivation of the adjacent Na+ channels as they become accomodated.
What are minature end-plate potentials?
They are small depolarisations (1mV) of the sarcolemma caused by the spontaneous release of vesicles (about 1 per second). These depolarisations are too small to trigger an muscle AP.
What is Myasthenia gravis?
An autoimmune disease targeting nACh receptors that causes patients to have profound muscle weakness which increases during exercise.
What is the mechanism of action of myasthenia gravis?
Antibodies directed against nAChR on postsynaptic membrane of skeletal muscle causes loss of function nAChR by complement mediated lysis and receptor degradation. Endplate potentials are reduced in amplitude leading to muscle weakness and fatigue.
What would happen to the minature end plate potenitals of an individuals suffering myasthenia gravis?
They would decrease in amplitue because there are less nAChR available.
What are depolarising blockers, such as succinylcholine, used for?
They are used in conjunction with general anaesthetic during surgical operations to prevent patients moving.
What is the difference between nicotinic and muscarinic receptors?
nAChR produce a faster depolarisation becuase it is an intrinsic ion channel (ligand-gated ion channel) in comparison mAChR produces a slower response because it is coupled to G-proteins which trigger a cascade of events in a cell.
Where are mAChR found?
On target tissues in which ACh release is activated by the parasympathetic branch of the autonomic system.
Where are nAChR found?
On skeletal muscle. They are activated by motor neurones.
List some functions that Ca2+ is responsible for or regulates:
Fertilisation - wave of Ca2+ when sperm meets egg
Proliferation, secretion (e.g. of hormones), neurotransmission, metabolism, contraction, learning and memory, apoptosis and necrosis
There is a huge gradient (10,000x) between [Ca2+]o and [Ca2+]i. What is the advantages of this gradient?
- Changes in [Ca2+]i occur rapidly with movement of little Ca2+
- Little Ca2+ has to be removed to re-establish resting conditions
There is a huge gradient (10,000x) between [Ca2+]o and [Ca2+]i. What is the disadvantages of this gradient?
- Energy expensive
- Inability to deal with Ca2+ easily leads to Ca2+ overload, loss of regulation and cell death (if things start to go wrong they spiral out of control quickly)
How is the gradient of Ca2+ set up and maintained?
- Relative impermeability of plasma membrane to Ca2+
- Expulsion of Ca2+ across the PM - two main mechanisms:
(i) Ca2+-ATPase
(ii) Na+/Ca2+ exchanger - Ca2+ buffers
The plasma membrane is relatively impermeable to Ca2+. How is this permebility regulated?
By the open/close state of ion channels.
How does Ca2+-ATPase respond to an increase in [Ca2+]i?
- [Ca2+]i increases
- Ca2+ binds to calmodulin (a Ca2+ binding protein) causing a conformational change
- Calmodulin binds Ca2+-ATPase
- Ca2+-ATPase removes Ca2+
What is the affinity and capacity of Ca2+-ATPase?
High affinity, low capacity - therefore can bind Ca2+ even at low concentrations but can only move across the PM small amounts of Ca2+.
What gradients does the Na+/Ca2+ exchanger use as a driving force?
Na+ flowing down its concentration gradient and electrical gradient into the cell. It therefore requires Na+K+-ATPase to set this up.
The Na+/Ca2+ exchanger is electrogenic. What does this mean?
It means it produces a change in the membrane potential of the cell - slightly depolarising the RMP (works best at RMP).
What type of protein channel is the Na+/Ca2+ exchanger?
Antiporter - secondary active transport (doesn’t require energy but uses a gradient that is set up by Na+K+-ATPase which does require energy.
What is the affinity and capacity of Na+/Ca2+ exchanger?
Lower afinity, higher capacity than Ca2+-ATPase
Why does Ca2+ diffuse more slowly than predicted from its ionic or hydrated radius?
Due to Ca2+ buffers (ATP and Ca2+ binding proteins).
List some Ca2+ binding proteins:
Parvalbumin, calreticulin, calbindin and calsequestrin.
What does Ca2+ diffusion depend upon?
- Concentration of Ca2+ binding molecules
2. Level of saturation of Ca2+ binding molecules
What are trigger proteins? How do they differ from Ca2+ buffers?
These are proteins which when Ca2+ binds have altered functions (either directly or by changing where the protein is in the cell). These are distinct from Ca2+ buffers that have the role of regulating free [Ca2+].
How does Ca2+ affect the trigger protein - synaptotagmin?
Binding of Ca2+ changes its shape and leads to the movement of ACh vesicles.
How does Ca2+ affect the trigger protein - troponin?
Ca2+ binding alters its shape and causes tropomyosin to shift and uncover the myosin head binding site on actin.
What are the two broad categories of Ca2+stores?
- Rapidly releasable
2. Non-rapidly releasable
What are the two main pathways that raise [Ca2+]i?
- Influx across PM
2. Influx from SER - intracellular store
How does Ca2+ influx across the plasma membrane?
- Through voltage-operated Ca2+ channels (VOCC)
2. Ionotropic receptors (ligand-gated channels)
How does the VOCC differ from cell to cell? What is the driving force that opens them and causes Ca2+ influx?
Different types of Ca2+ channels with different regulatory features are found in different cell types (e.g. L,N,R,P/Q,T-types). They are opened when depolarisation of the membrane changes the charge of their protein side chains, causing a conformation change that opens the channel. Once open Ca2+ influx is driven by the Ca2+ concentration gradient.
What is the mechansims of action of ionotropic receptors in Ca2+ influx?
Binding of ligands/agonist to these receptors causes a conformation change that opens the channel. Ca2+ influxes into the cell down its concentration gradient (+/- electrical gradient).
What are some examples of ligands and their ionotropic receptors?
- NMDA/AMPA receptors for glutamate
2. NACh receptors for acetylcholine
What is an ionotropic effect?
An ionotropic effect can be applied to the effect of a transmitter substance or hormone on its target. The transmitter or hormone activates or deactivates ionotropic receptors (ligand-gated ion channels).
What is the general mechanism by which G-protein coupled receptors work?
- Activated by binding of a ligand
- Ligand binding changes conformation allowing G-protin to bind to intracellular component
- G-protein becomes activated and diffuses around cell activating/deactivating other molecules
How are many of the cell responses to GPCRs mediated?
By changing [Ca2+]
How many different types of GPCRs are there?
Over 800
How many GPCRs are the targets for drugs?
~50
What percentage of drugs work by targeting GPCRs?
30-50%
Give some examples of stimuli for GPCRs:
- Hormones
- Neurotransmitters
- Ions
- Odourants
- Taste
How can GPCRs affect the release of Ca2+ from intracellular stores?
Gq proteins:
- Alpha-q activates PLC
- PLC hydrolyses PIP2 -> IP3 and DAG
- IP3 is released into cytosol and activates IP3 receptor (ligand-gated ion channel) in SER membrane -> opens and Ca2+ efflux into cytosol
- DAG remains bound to membrane and along with Ca2+ activates PKC which then goes on to phosphorylate other molecules
What is a major role of the ligand glutamate?
It is a major excitatory neurotransmitter in the brain.
The ryanodine receptor is structurally similar to the IP3 receptor, but how does it differ?
It does not open in response to IP3 but in response to Ca2+ = Ca2+-induced Ca2+ release (CICR).
In which cells is CICR particularly important?
Cardiac myocytes - Ca2+ influx through VOCC activates ryanodine receptors
How does activation of ryanodine receptors differ between cardiac and skeletal muscle?
In skeletal muscle conformational changes to the L-type Ca2+ channels (VOCC) is passed on to the ryanodine receptors. In cardiac muscle it is Ca2+ binding that causes a conformational change to the ryandine receptors.
Explain how depolarisation of a T-tubule in a cardiac myocyte results in contraction of the muscle:
- Sarcolemma depolarises down T tubule - via V-gated Na+ channels
- VOCC are activated by local depolarisation
- Influx of extracellular Ca2+ (15%)
- Ca2+ in cytosol ativates RyR
- Influx of Ca2+ from SER stores (85%) directly next to contractile machinery
- Ca2+ binds to troponin C and causes a conformation change that allows the mysoin head to bind and contraction to occur.
In the cardiac myocyte what is the major and minor proteins that restore the intracellular Ca2+ to its resting levels?
Major - SERCA (SER Ca2+-ATPase)
Minor - Na+Ca2+-exchanger
Why does the action of Na+/Ca2+ reverse in stage 2 of a cardiac action potential?
The high concentration of Na+ in membrane microdomains during a cardiac action potential can result in a transient reversal of Na+ concentration gradient and therefore a transient reverseal of NCX, causing it to exchange Ca2+ from outside to inside the cell.
Name a non-rapidly releasable store of Ca2+:
mitochondria
What affinity and capacity does the mitochondrial Ca2+ uptake uniporter have?
Low affinity, high capacity
What is the role of mitochondrial Ca2+ uptake?
Important in neurones perhaps less so in other cells.
- Ca2+ buffering - regulate pattern and extent of Ca2+ signalling
- Stimulation of mitochondrial metabolism - match energy demand with supply
- Role in cell death - e.g. apoptosis
What is the role of Ca2+ and mitochondria in cell death?
Mitochondrial calcium overload/ dysfunction key for triggering cells death in e.g. ischemia and traumatic brain injury, ALzheimer’s, Parkinson’s, Huntington’s and amyotrophic lateral sclerosis.
Why does [Ca2+]i have to return to its basal state?
Too much Ca2+ for too long is toxic and leads to cell death.
What three things have to happen for [Ca2+]i to return to its basal state?
- Termination of signal (desensitisation/ ligand removal)
- Ca2+ removal
- Ca2+ store refilling
How are Ca2+ stores refilled?
- Recycling of released cytosolic Ca2+ e.g. cardiac myocyte
2. VOCC and/ or capacitative Ca2+ entry
Refilling stores by capacitative Ca2+ entry, happens in which cells?
Non-excitable cells (also in excitable cells).
What is the mechanism behind capacitative or store-operated channels (SOC)?
SOC channels are activated in response to a ‘depleted’ signal from SER and cause an influx of Ca2+ across the PM.
