Calcium Sensitisation

Recall and integrate the roles of Ca²⁺, calmodulin, MLCK and myosin RLC in activation of smooth muscle.

As mentioned here, Ca²⁺-calmodulin complexes can activate myosin light chain kinase (MLCK). MLCK can then phosphorylate serine 19 on the myosin regulatory light chain (RLC). This then allows it to interact with actin, which is important for contraction.

Another important molecule to take note of is MLCP, or myosin light chain phosphatase. This reverses the action of MLCK. Relative amounts of MLCP and MLCK are important in determining whether the cell is contracting or relaxing. I'll get to MLCP in a bit.

Explain the evidence for a role of Ca²⁺ and calmodulin in contraction.

We know that there is a role of Ca²⁺ as skinned fibres (i.e. muscle fibres with all of their lipid content removed- including membranes) only display contraction when calcium levels are high enough. We also know that there is a role of calmodulin in contraction because increased amounts of calmodulin, at the same concentration of Ca²⁺, can cause greater amounts of contraction.

Define calcium sensitisation and give evidence for its existence.

Calcium sensitisation refers to the generation of relatively large amounts of force from relatively small amounts of calcium. Some of the evidence for this comes from experiments done with calcium-binding dyes. Depolarisation-induced contractions show high levels of intracellular calcium that last for a while, whereas agonist-induced contractions show high levels of intracellular calcium that quickly drop off without having any deleterious effect on the contraction. Other studies have shown that agonist-induced contractions can display the same amount of force or greater than a depolarisation-induced contraction at the same concentration of calcium. These results, taken together, suggest a sensitisation effect: the cell becomes more "sensitised" to calcium so that less is required to have the same effect.

Explain how calcium sensitisation is produced through the rho-ROK and PKC pathways.

Rho is a G-protein, which refers to the fact that it binds GTP (it is NOT related to the trimeric G-proteins that I've covered when talking about G-protein coupled receptors). Rho activates Rho Kinase (ROK), which phosphorylates and inhibits MLCP. Hence, Rho makes it more likely that MLCK will overpower MLCP, maintaining contraction even at lower amounts of Ca²⁺.

A second possible method of sensitisation occurs through the PKC pathway. As I've mentioned before, phosphatidylinositol breaks up into IP₃ and diacylglycerol (DAG). I've already spoken about the effects of IP₃ in smooth muscle here, so let's talk about DAG. DAG can activate protein kinase C (PKC), which also phosphorylates and inhibits MLCP. PKC can also phosphorylate another protein called CPI-17, which also inhibits MLCP. CPI-17's method of inactivating MLCP is slightly different: it does its job by binding to MLCP's active site (rather than phosphorylating it).

Recall and explain the functions of rho, ROK, MLCP, DAG, PKC and CPI-17 in smooth muscle contraction

This is basically just a summary of the points I've touched on in this post:

• Rho- G-protein that activates ROK.
• ROK (Rho Kinase)- phosphorylates and inhibits MLCP.
• MLCP (Myosin Light Chain Phosphatase)- dephosphorylates myosin, stopping contraction.
• DAG (diacylglycerol)- activates protein kinase C.
• PKC (protein kinase C)- phosphorylates and inhibits MLCP. Also phosphorylates and activates CPI-17.
• CPI-17- inhibits MLCP by binding to its active site.