Calcium Regulation Tool Box

Question 1

Who discovered that extracellular calcium was needed?
What preparation did he use?
Results?

Answer 1

Ringer, 1883
Discovered that extracellular Ca2+ is necessary for myocardial contraction using a frog’s heart perfused with blood mixture which was then replaced with saline. Amplitude of contractions declined with saline and were recovered by calcium chloride.
Led to an understanding of excitation-contraction coupling and processes involved with it.

Question 2

What happened upon withdrawal of extracellular calcium? In skeletal and cardiac muscle?

Answer 2

Withdrawal of extracellular Ca2+ abolishes contraction of cardiac muscle, but not in skeletal muscle as they do not depend on Ca2+ entry into the cell.

Question 3

What are the types of sarcolemmal Ca channels? Which is dominant? Where is the other present mostly?

Answer 3

Two types of sarcolemmal Ca channels in cardiac myocytes (T - and L-type). The current carried by the L-type Ca2+ channel (ICa,L) is dominant and is present in all cardiac myocytes.
In many species, ICa,T is present to a variable extent in atrial and conducting system cells (e.g. Purkinje fibers).

Question 4

What evidence is there for the properties of the calcium currents in cardiac cells?
Activation and inactivation?
T-type calcium channel location?
What happens during plateau phase?

Answer 4

Hirano et al., 1989
Two types of Ca2+ currents were recorded in single canine Purkinje cells using a whole cell voltage clamp technique.
T-type current was easily separated from L-type current, because its voltage dependence of inactivation and activation was more negative and it decayed rapidly.
Found that Rabbit ventricle lacks ICa,T
During the ‘plateau phase’ of the AP in ventricular cells, Ca2+ enters the cell (mostly) via voltage gated L-type Ca2+ channels but this is not sufficient to activate the myofilament directly

Question 5

What happens after calcium entry?
When is the current sustained and what counterbalances it?
Can it be modified by anything?

Answer 5

Ca2+ entry triggers further Ca2+ release by activation of closely opposed RyR Ca2+ channels located in the junctional SR (i.e. next to t-tubule) – CICR
In ventricular cells, ICa,L is sustained during the plateau phase of the ventricular AP and is the primary inward current at this period (counterbalanced by K currents).

Question 6

What is the structure of the calcium L-type current?

Answer 6

Main α1 subunit has 4 domains, each with 6 transmembrane spans (S1-S6)
The L-type Ca2+ channel antagonists bind to IIIS5, IIIS6 and IVS6 regions.
There is a possible calmodulin binding site (cytosolic, Ca2+-dependent regulatory protein) and intracellular phosphorylation sites.

Question 7

What evidence is there for the minimujm structure needed for current to pass?

Answer 7

Perez-Reyes et al., 1989
Transfection into mouse L-cells of DNA cloning for the alpha 1 subunit which do not usually express other subunits was sufficient to produce Em dependent ICa and is very similar in structure to the Na channel.
Suggests that this subunit is the minimum structure of this channel.

Question 8

What is the stoichiometry of the NCX channel?

Answer 8

Stoichiometry of the sodium calcium exchanger is 3Na to 1Ca, such that 1 positive charge is moved in the direction of Na transport.
Thus Ca influx via Na/Ca exchange produces an outward INa/Ca and Ca extrusion causes inward INa/Ca.

Question 9

What does the direction and amplitude of the ɴCX current depend on?

Answer 9

the membrane potential (Em) as well as the intra- and extracellular Na+ and Ca2+ concentrations

Question 10

Under normal conditions what is the reversal potential of ɴCX? and what current occurs at the resting membrane potential?

Answer 10

Under normal conditions the reversal potential (ENa/Ca) is about -40 mV, and inward INa/Ca occurs (Ca extrusion) at the resting potential Em -80 mV

Question 11

When does the current reverse and what does that do to the calcium?

Answer 11

Upon depolarization to the AP peak, Em passes through ENa/Ca and INa/Ca reverses and becomes outward (Ca influx)

Question 12

Can NCX reverse mode trigger SR Ca2+ release?

What evidence is there for or against it?

Answer 12

Na/Ca exchanger can allow some Ca2+ to enter the cell during the action potential.

Sipido et al., 1997
In guinea pig ventricular myocytes, whole-cell voltage clamp and fluorescence Ca2+ indicators compared the characteristics of Ca2+ release triggered by reverse-mode ɴCX and by L-type Ca2+ current (ICaL) during depolarizing steps.
With Ca2+ entry through Na+-Ca2+exchange only, Ca2+ release from the SR is decreased, directly related to the amplitude of the depolarizing step and has significant delay related to Ca2+ entry through the exchanger.
With both ICaL and reverse-mode Na+-Ca2+exchange present, Ca2+ release is triggered by ICaL, and a contribution of reverse-mode Na+-Ca2+ exchange to the trigger could not be detected at potentials below +60 mV.
Its role as a trigger for Ca2+release during the action potential is likely to be negligible.
This differs from the results by Levi et al. (1994) but the major difference is the use of a steady-state block of ICaL compared to ʟevys rapid switch device. The advantage of steady state block is that one can obtain a more complete block of ICaL

Question 13

Give the components of the calcium SR regulation

Answer 13

Sarcolemmel Ca pump
SERCA
Phospholamban
RyR release channel

Question 14

What is the mechanism of the sarcolemmel Ca2+ pump?
What is the contribution to Ca removal?
What contributes most?

Answer 14

Transfers energy from ATP to a high energy phosphorylated intermediate
This energy is then used to transfer Ca2+ against its concentration gradient out of the cell. 1 Ca2+ per ATP. It has a similar structure to SR Ca ATPase channel
Under normal circumstances the SL-Ca-pump makes only a small contribution to the removal of Ca2+ from the cytosol
Most of the Ca2+ is extruded from cell via the Na/Ca exchanger or re-accumulated by the sarcoplasmic reticulum (SR) via the SR Ca-ATPase (SERCA)

Question 15

What is SERCA and what evidence is there for it’s role in calcium uptake?

Answer 15

Ebashi (1961)
- identified the membrane associated “relaxing factor” in muscle responsible for lowering cytoplasmic [Ca]
The sarco-(endo)-plasmic reticulum Ca-ATPase (SERCA) is a member of the ATPase family
The transport starts with two Ca ions and one ATP binding to high affinity binding sites, the terminal ATP is transferred to Aspartame-351 on the Ca pump.

Clarke et al., 1989
Used oligo-nucleotide-directed mutagenesis to identify the amino acids involved in Ca2+ binding
Showed Ca binds to M4-6 and M8 which may form a channel and phosphorylation decreases affinity for calcium to be released into SR lumen

Question 16

What is phospholamban and what does it do?

What evidence is there for it’s role in preventing Ca uptake via SERCA

Answer 16

PLB is an endogenous inhibitor of SERCA in cardiac and slow twitch skeletal muscle.
The main mechanism by which catecholamines accelerate relaxation (Lusitropy) in of the heart.

Sham et al., 1991
A monoclonal antibody of PʟB to interfere with interaction between PLB and SERCA in intact whole cell patch clamped guinea pig ventricular myocytes.
Adding the antibody enhanced the rate of uptake of Ca2+ and significantly suppressed the ability of isoproterenol to enhance the rate of uptake.
The stimulatory effect of the antibody could be mimicked by the catalytic subunit of cAMP. Suggests may be the mechanism of catecholamine-enhanced rate of Ca2+ uptake.

Question 17

What channel does calcium get released from the SR?
What is its structure and what binding sites are associated with it?
Where are they located?

Answer 17

‘ryanodine receptor’ (RyR), because the plant alkaloid ‘ryanodine’ binds to the channel with high affinity and alters its properties
RyR2, which is found in cardiac muscle, it is a homotetramer: i.e. four identical subunits combined to form a functional channel.
RyR2 has cytosolic binding sites associated with calcium activation and inactivation, calmodulin and FKBP.
Physiologically, RyRs are arranged in the junctional regions of the SR, in close association with the L-type Ca2+ channels present in the t-tubules

Question 18

What evidence is there for the structure of the RyR channel? What is it activated by?

Answer 18

Serysheva et al., 1999
Using electron cryomicroscopy and angular reconstitution, determined the 3D structure of the skeletal muscle RyR channel is activated by analogue of ATP in the presence of Ca.
Initial work but further studies of the 3D architecture of the SR Ca release channel at higher resolution to know mechanism.

Question 19

Give the papers for:
Extracellular Calcium
Two calcium sarcolemmel channels
Minimum structure for Ical
Can reverse mode NCX cause CICR?
SERCA mechanism (2)
PLB
RyR

Answer 19

Extracellular Calcium
- Sidney Ringer, 1883
Two calcium sarcolemmel channels
- Hirano 1989
Minimum structure for Ical
- Peres-Reyes 1989
Can reverse mode NCX cause CICR?
- Sipido 1997
SERCA mechanism
- Ebashi 1961
- Clarke 1989
PLB
- Sham 1991
RyR
Serysheva 1999