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Describe how action potentials open Ca2+ channels in cell membranes

An AP arriving at the presynaptic membrane causes the opening of voltage-gated Ca2+ channels which allows an influx of Ca2+ down their concentration gradient into the nerve terminal.

This increase in  intracellular Ca2+ concentration is significant despite the intracellular Ca2+ concentration remaining low to everything else.

Influx of Ca2+ causes the release of the neurotransmitter (via exocytosis)

1

Describe some aspects of the diversity of Ca2+ channels

Very similar alpha-unit subunit structure to voltage gated Na+ channels include a voltage sensor region and a pore-forming domain which is quite selective for Ca2+.

But Ca2+ channels have structural diversity - a blocker that blocks one calcium channel will not necessarily block another.

Different Calcium channels have different primary locations so selectively blocking one type of channel can have a localised effect.

2

Give some specific examples of Ca2+ channel blockers

DHP = Dihydropyridines Nifedipine is used to treat a variety of clinical conditions e.g. High blood pressure

As well as functional alpha subunits, other subunits can co-assemble with the alpha-subunit, modifying and moderation the channel e.g. protein kinase A increases the amount of open Ca2+ channel so there is increased influx of Ca2+.

Phosphorylation sites are a way of regulating channel activity.

3

What is Fast Synaptic Transmission?

Receptor protein is also an ion channel

Binding of the transmitter causes the channel to open

4

How does Ca2+ trigger a release of neurotransmitter?

Many cellular processes are dependent on a change in intracellular Ca2+.

The channels are located close to vesicle release sites.

The increase in intracellular Ca2+ following an AP reaching the motor nerve terminal triggers ACh release.

Ca2+ binds to Synaptotagmin and vesicle is brought close to the membrane.

The Snare Complex forms and makes a fusion pore through which ACh is released.

ACh binds and activates nicotinic ACh receptor channels on the post-junctional membrane to produce an end-plate potential; this depolarisation in turn raises the muscle above threshold so that an action potential is produced in the muscle membrane.

5

Describe the selectivity of the nicotinic ACh receptor

Ligand-gated ion channels which are selective for cations - for Na+ and K+

Consequently the membrane potential reaches ~halfway between E(Na) and E(K)

6

Describe the sequence of the events during neuromuscular transmission

Brief depolarization (the end-plate potential) caused by activation of nAChR by ACh binding will activate adjacent Na+ channels due to local spread of charge (depolarization of adjacent muscle membrane) thereby initiating an AP in the muscle fibre.

Muscle fibre then contracts due to excitation-contraction coupling.

ACh is degraded quickly by ACh esterase in the the synaptic cleft.

7

How do Competitive Blockers work?

Bind at the molecular recognition site for ACh, closing the the nicotinic ACh receptor, preventing ACh from binding.

Competitive blocker drugs e.g. Tubocurarine

This causes paralysis

8

Describe how Depolarizing Blockers work

E.g. Succinylcholine is used in operations to induce paralysis.

Succinylcholine will cause a maintained depolarization at the post-junctional membrane so adjacent Na+ channels will fail to be activated due to accommodation (they have also become inactivated).

Therefore a muscle AP is not generated because there is no local spread of charge.

Miniature end-plate potentials are not enough to generate an AP.

9

What is Myasthenia Gravis?

Autoimmune disease targeting nACh Receptors

Patients suffer profound weakness which increases with exercise, drooping eyelids

It is caused by antibodies directed against nACh receptors on post-synaptic membrane of skeletal muscle .

End-plate potentials are reduced in amplitude leading to muscle weakness and fatigue.

Each quantum of ACh released produces a smaller response than in normal muscle because there are less nACh receptors available.

Amount of ACh released by each vesicle is still the same

10

How could you treat Myasthenia Gravis?

Could alleviate symptoms by treating with ACh esterase inhibitors to increase the amount of time ACh is in the synaptic cleft - preventing rapid breakdown.

11

Explain how nicotinic and muscarinic ACh receptors operate differently

nAChR produces a fast depolarization because it is a ligand gated ion channel.

mAChR produces a slower response because they are coupled to G-proteins which trigger a cascade of events in the cell.

12

Explain the importance of control of intracellular Ca2* concentration

It is critical for normal cellular activity and for pathophysiological changes in cell function.

Changes in intracellular Ca2+ are responsible for or regulate:

Fertilization

Proliferation

Secretion

Neurotransmission

Metabolism

Contraction

Learning and memory

Apoptosis and necrosis

13

Describe the intracellular Ca2+ concentration and its significance

Under resting/basal conditions, intracellular [Ca2+] is maintained at a very low level compared to [Ca2+] in extracellular fluid.

For changes in intracellular [Ca2+] to be used as a signalling event, the intracellular [Ca2+] must also be rapidly restored to basal levels.

Elevations of intracellular [Ca2+] that are too great or occur for too long are detrimental to the health of the cell, emphasising the need to tightly control intracellular [Ca2+].

As Ca2+ cannot be metabolised! the cell has to regulate intracellular Ca2+ concentration based largely on moving Ca2+ into and out of the cytoplasm.

14

What are the advantages and disadvantages of the Ca2+ concentration gradient?

Extracellular [Ca2+]: 1-2mM (10x-3 M)

Intracellular [Ca2+]: 100nM (10x-7 M)

Advantages: changes in intracellular [Ca2+] occur rapidly with little movement Ca2+

Disadvantages: Ca2+ overload leads to loss of regulation and cell death. There is a large inward gradient which is very energy expensive - requires a lot of energy to maintain this ionic gradient (using ATP hydrolysis)

15

What mechanisms does the Ca2+ gradient rely on?

1. relative impermeability of the plasma membrane

2. cell's ability to expel Ca2+ across the plasma membrane

3. Ca2+ buffers

4. Intracellular Ca2+ stores - Rapidly Releasable and Non-rapidly Releasable

16

Explain how the relative impermeability of the plasma membrane is important in maintaining the Ca2+ concentration gradient

Allows the cell to keep the intracellular [Ca2+] low.

Membrane permeability is regulated by open/closed state of ion channels.

17

Explain how the ability of the cell to expel Ca2+ across the plasma membrane is important in maintaining the Ca2+ concentration gradient

A) Energy dependent PMCA

B) Na+/Ca2+ Exchanger

18

Explain how PMCA is important in maintaining Ca2+ concentration gradient

feedback mechanism

When intracellular [Ca2+] increases, Ca2+ binds to calmodulin (a trigger protein).

This complex binds and stimulates Ca2+-ATPase which removes Ca2+ from the cell.

Considered high affinity, low capacity when intracellular [Ca2+] is returning back to normal levels the pump makes resting level of [Ca2+]i is reached properly

19

Explain how Na+/Ca2+ Exchanger is important in maintaining Ca2+ concentration gradient

Na+ gradient is used as a driving force (requires Na+ pump)

Antiporter is electrogenic - works best at removing Ca2+ at resting membrane potential

Transports 3Na+ into the cell per 1 Ca2+ out

Considered low affinity, high capacity - works best when [Ca2+]i is high, good at removing large amounts of Ca2+.

May be important in some cell types for Ca2+ removal or contribution to changes in membrane potential.

20

Explain how Ca2+ buffers are important in maintaining Ca2+ concentration gradient

 

Ca2+ buffers limit diffusion, through ATP and Ca2+-binding proteins such as parvalbumin, calreticulin, calbindin, calsequestrin.

These binding proteins have no other function.

Many other proteins bind Ca2+ which alters their function -"trigger" proteins - e.g Synaptotagmin

Ca2+ diffusion depends on concentration of binding molecules and their level of saturation.

21

How is intracellular [Ca2+] elevated?

In most cells, basal [Ca2+]i is ~100nm.

This can rise to ~1 micromole when Ca2+ is being used to regulate cellular activity.

The mechanisms for change in [Ca2+]i are:

1. Ca2+ influx across the plasma membrane I.e. Altered membrane permeability

2. Ca2+ release from rapidly releasable intracellular stores

3. Ca2+ release from non-rapidly releasable intracellular stores.

22

Describe how Ca2+ influx across the plasma (I.e. Altered membrane permeability) can elevate intracellular [Ca2+]

A) Voltage-Operated/Gated Ca2+ channels: depolarisation causes a conformational change which opens the channel and allows influx of Ca2+ down concentration gradient.

B) Receptor-Operated Ca2+ channels: ligand/agonist causes a conformational change opening the channel and allows Ca2+ to enter down its concentration gradient.

23

Describe the rapidly releasable intracellular stores

stores of Ca2+ are set up inside sarco/endoplasmic reticulum by the SERCA (Sarco/Endoplasmic Reticulum Ca2+ ATPase).

Ca2+ is moved in using the energy from ATP hydrolysis and binding to proteins such as calsequestrin.

This process is not regulated by Calmodulin.

24

What are the mechanisms involved in how Ca2+ is released from rapidly-releasable stores?

G-Protein Coupled Receptors

Ca2+-induced Ca2+ Release (CICR)

25

How are GPCRs involved in Ca2+ release from rapidly releasable stores?

A ligand binds to the GPCR on the cell membrane, activating its G-alpha-q subunit.

This binds to the phospholipid PIP(2), releasing IP(3) which in turn binds to its receptor (ligand-gated ion channels) on the sarco(endo)plasmic reticulum, triggering the release of calcium down its concentration gradient.

Stimulus for GCPR signalling: hormones,neurotransmitters, ions, odourants, taste

note: heterotrimeric G-protein has 7 transmembrane domains

26

How does Ca2+-induced Ca2+ Release (CICR) work?

Ca2+ binds to the Ryanodine receptor on the side of the sarco(endo)plasmic reticulum, triggering the release of Ca2+ into the cell down its concentration gradient.

27

What is an important physiological role for CICR?

In the cardiac myocyte, Ca2+ entry through VOCCs due to the depolarisation of the membrane results in an explosive release of large amount of Ca2+ from the intracellular stores as the influx of Ca2+ act on Ryanodine receptors of the sarcoplasmic reticulum to drive Ca2+ release from this intracellular store.

This explosive release of Ca2+ around the contractile proteins ensures a strong and coordinated response.

28

Describe Ca2+ handling by cardiac myocytes

At the height of depolarisation, conditions will favour reversal of the Na+/Ca2+ exchanger which result in a small amount of Ca2+ entry.

As [Ca2+]i increases and membrane repolarization starts, NCX will revert back to Ca2+ extrusion to lower [Ca2+]i and Ca2+ will also be pumped back into the SR by the SERCA in preparation for another release event.

Ca2+ channels show voltage-sensitive activation and inactivation similar to Na+ channels but much slower.

This, along with low K+ conductance at depolarised potentials, allows prolongation of the depolarisation in cardiac cells.

29

What is the role of Ca2+ in contraction?

Ca2+ binds to troponin which undergoes a conformational change causing Tropomyosin to move and reveal binding sites on actin for the myosin head groups

In the presence of ATP, myosin undergoes cycles of attachment and detachment that, coupled with a movement of the head group, results in a sliding of the actin along the myosin bundles and a shortening (contraction) of the myocyte.