M&R 5.2 Control of intracellular Ca2+

Question 1

Basal Ca2+ levels inside the cell are ~10,000 fold lower than the outside. Name 2 advantages and 2 disavantages of this

Answer 1

Advantages

1. Inward movement of little Ca2+ causes rapid changes in [Ca2+]i
2. Little Ca2+ has to be removed to re-establish resting conditions

Disadvantages

1. Energy expensive to maintain
2. Any inability to deal with Ca2+ can easily lead to Ca2+ overload and cell death

Question 2

Name 4 factors that maintaining the Ca2+ gradient relies on

Answer 2

1. relative impermeability of the plasma membrane
2. Ability to expel Ca2+ across membrane (using Ca2+ ATPase and Na+ - Ca2+ exchanger)
3. Ca2+ buffers
4. Intracellular Ca2+ stores (both rapidly and non-rapidly releasable)

Question 3

What regulates the membrane permeability to Ca2+?

Answer 3

The open/closed state of ion channels

Question 4

How does the Ca2+ ATPase extrude calcium from the cell?

Answer 4

[Ca2+]i increases
Ca2+ binds to calmodulin
Ca2+-calmodulin complex binds Ca2+-ATPase
Ca2+-ATPase removes Ca2+

Question 5

The Ca2+-ATPase has ______ affinity and ______ capacity

Answer 5

High affinity (can bind Ca2+ even when conc is low)

Low capacity (can’t remove lots of Ca2+ this way - better for when near basal levels to get last bits out)

Question 6

How does the Na+/Ca2+ exchanger extrude calcium from the cell?

Answer 6

Antiporter
Uses inward concentration gradient of Na+ to move 3Na+ into cell in exchange for 1 Ca2+ out
Works best at RMP (because inside cell is -ve so Na+ is moving down both its electrical gradient as well at its conc gradient )
Less active in a depolarised membrane (because Na is moving down its conc gradient but up its electrical gradient)

Question 7

The Na+/Ca2+ exchanger has _______ affinity and ________ capacity

Answer 7

Low affinity
High capacity
(so more helpful for initial process of moving lots of Ca2+ out)

Question 8

What do Ca2+ buffers do?

Answer 8

Bind to Ca2+ to limit its diffusion (Ca2+ diffuses more slowly than would otherwise be predicted)
Therefore can help to buffer any changes in Ca2+

Question 9

Name some Ca2+ buffers

Answer 9

Calsequestrin
Calbindin
Parvalbumin
Calreticulin

Question 10

How are Ca2+ trigger proteins different from Ca2+ buffer proteins? List some examples.

Answer 10

Buffer proteins bind Ca2+ to buffer changes in [Ca2+]
Trigger proteins are proteins which bind Ca2+ to alter their own activity, e.g:
synaptotagmin (Ca2+ sensor involved in NT release)
calmodulin (activates Ca2+ ATPase in plasma membrane)
troponin (in muscle)

Question 11

[Ca2+]i is elevated by which 3 main routes?

Answer 11

1. Ca2+ influx across membrane
2. Ca2+ release from ‘rapidly-releasable’ stores
   - -> GPCRs
   - -> Ca2+ induced Ca2+ release (CICR)
3. Ca2+ release from non-rapidly releasable stores (mitochondria)

Question 12

By which 2 mechanisms can Ca2+ cross the plasma membrane?

Answer 12

1. Voltage-gated Ca2+ channels (VGCCs)

2. Ionotropic receptors (ligand gated ion channels with selectivity for Ca2+ ions)

Question 13

Name some examples of ionotropic (ligand-gated) Ca2+ channels

Answer 13

NMDA/AMPA receptors for glutamate (glutamate is major excitatory NT in brain)

Some nAChRs (certain ones have a particular combination of subunits that make them permeable to Ca2+ too)

Question 14

Where is the rapidly-releasable intracellular store of calcium?

Answer 14

The endoplasmic reticulum (or sarcoplasmic reticulum in muscle)

Question 15

Describe Ca2+ storage in the ER/SER

Answer 15

Free Ca2+ in the ER/SER is greater than in the cytoplasm

Therefore an active uptake process is required (=SERCA - sarcoendoplasmic reticulum Ca2+ ATPase)

ER/SER can store lots of Ca2+ by binding it to proteins with low affinity but high capacity (e.g. calsequestrin - just allows the store to hold onto Ca2+ better than if it was free)

Ca2+ release can be regulated by ion channels (large outwards gradient so Ca2+ can rapidly enter cytoplasm)

Question 16

Name 2 methods by which Ca2+ release from rapidly-releasable stores is regulated, and which receptors are involved in each.

Answer 16

1. Via GPCRs (by activation of IP3 receptors)

2. Via calcium-induced calcium release [CICR] (by activation of ryanodine receptors)

Question 17

Describe GPCR mediated release of Ca2+ from rapidly releasable stores

Answer 17

Activation of G alpha q (Gq) G-protein via GPCR
Gq activates phospholipase C (PLC)
PLC cleaves a membrane lipid into DAG & IP3
IP3 acts on the IP3 receptor (a ligand-gated ion channel on the ER)
Binding of IP3 causes channel to open & Ca2+ to rush out of store down its conc gradient

(NB: DAG and increased [Ca2+]i as a result of IP3 also both work to activate protein kinase C)

Question 18

Describe release of Ca2+ from rapidly-releasable stores by calcium-induced calcium release (CICR)

Answer 18

Ca2+ enters cell from another source (e.g. VGCCs, ionotropic receptors, or from intracellular stores)
The Ca2+ activates Ryanodine receptors on the ER/SER
Conformational change - pore opens and Ca2+ rushes out into cytoplasm

Question 19

Where does CICR have a particularly important physiological role?

Answer 19

In cardiac myocytes

Question 20

Describe CICR in cardiac myocytes

Answer 20

T-tubule allows depolarisation to get deep into cell
Change in MP detected by VGCCs - open - Ca2+ rushes into cell down conc gradient
Ca2+ activates ryanodine receptors on the SER
= Ca2+ release from SER

Question 21

In cardiac myocytes, what proportion of the Ca2+ entering the cytoplasm comes from outside the cell and what proportion comes from intracellular stores?

Answer 21

15% comes from outside the cell (via VGCCs)

85% comes from intracellular stores (via ryanodine receptor RyR)

Question 22

What is the advantage of the majority of the calcium released into cardiac myocytes coming from intracellular stores?

Answer 22

It allows an explosive release of Ca2+ right next to the contractile machinery
This ensures a strong, co-ordinated contractile event

Question 23

How is cytoplasmic Ca2+ reduced again following a depolarisation?

Answer 23

1. As [Ca2+]i increases and repolarisation starts, the Na+/Ca2+ exchanger reverts to Ca2+ extrusion from cell, lowering [Ca2+]i
2. SERCA pumps Ca2+ back into the SR in preparation for another release event
   (most of what is released is recycled ready for the next beat]

Question 24

How does Ca2+ influx alter the cardiac myocyte AP compared to other APs?

Answer 24

VGCCs activate and inactivate similarly to Na+ channels but much slower
This means that once VGNCs have already been inactivated, VGCCs are still open and allowing Ca2+ influx
This allows more prolonged depolarisation and causes the long plateau of the cardiac myocyte AP

Question 25

What else has a small contribution to Ca2+ entry in the cardiac myocyte AP?

Answer 25

When the membrane depolarises, VGNCs open and Na+ enters the cell down its concentration gradient
The alteration of the Na+ gradient allows reversal of the Na+/Ca2+ exchanger (NAX), so that a small amount of Ca2+ enters rather than being extruded

Question 26

Describe the functional role of Ca2+ in muscle contraction

Answer 26

Increased [Ca2+]i in cytoplasm of myocyte
Ca2+ binds to troponin, initiating a conformation change
This causes tropmyosin to move and reveal actin binding sites
Myosin head groups can now bind to actin binding sites
Myosin undergoes cycles of attachment and detachment (using ATP) and head group moves, so that there is shortening of the sarcomere
=contraction of myocyte

Question 27

Where are the non-rapidly releasable intracellular stores of calcium?

Answer 27

In the mitochondria

Question 28

How do mitochondria uptake Ca2+?

Answer 28

Via a uniporter
Uses the driving force from respiratory chain proton production

(has low affinity but high capacity, so is better when cytoplasmic Ca2+ conc is high)

Question 29

Mitochondrial stores of Ca2+ seem to be most important in what type of cells?

Answer 29

Neurons

Question 30

Name 3 roles of mitochondrial Ca2+ stores

Answer 30

1. Ca2+ buffering (to regulate pattern/extent of Ca2+ signalling)
2. Stimulation of mitochondrial metabolism (to match energy demand with supply)
3. Cell death (mitochondrial Ca2+ overload is key in triggering cell death)

Question 31

Ca2+ stores can be refilled by:

Answer 31

1. Recycling of released Ca2+ from cytosol back to store (especially in cardiac myocytes - other cells find it easier to send Ca2+ out across plasma membrane before they refill)
2. Capacitive Ca2+ entry (particularly in non-excitable cells)

Question 32

Describe capacitive Ca2+ entry

Answer 32

Store-operated channel (SOC) located on plasma membrane
When ER is depleted of Ca2+ a ‘depleted’ signal is sent to SOC
Ca2+ enters the cell via SOC
Once in the cell, the Ca2+ can enter the ER via SERCA