Excitation Contraction Coupling

Second post on smooth muscle! In this post, you'll probably start to appreciate how weird smooth muscles are. (See here for my first post on smooth muscle.)

Describe how agonist binding to P_2X receptors or other receptors (e.g. M-cholinergic and α-adrenergic) produces contraction through electromechanical coupling using ROC and VOC.

Okay, I guess I should start by explaining some of the terminology. Electromechanical coupling is, in a nutshell, coupling of something electric (in this case the membrane potential) with something mechanical (contraction). ROCs are receptor-operated channels (which is basically a fancy name for a ligand-gated ion channel) and VOCs are voltage-operated channels (i.e. voltage-gated channels).

For some reason this lecture contains an example of how this works in skeletal muscle, which isn't really that relevant now, but will probably become relevant later when we move onto learning about skeletal muscle. In skeletal muscle, ACh stimulates N-cholinergic (i.e. nicotinic) receptors, which are non-selective ROCs. They allow both Na⁺ and K⁺ to pass through, causing depolarisation. Depolarisation then opens Na⁺ VOCs.

A similar principle occurs in smooth muscle, but with different receptors. In smooth muscle, ATP activates P_2X receptors, which are non-selective ROCs that allow the passage of Na⁺, Ca²⁺ and K⁺. Opening of these channels causes depolarisation, which opens L-type Ca²⁺ channels (which are VOCs).

P_2X receptors are possibly the only "true" ROCs in smooth muscle- there are other ROCs, but they seem to require intermediate steps. One example of this is the ACh ROC. ACh does not actually bind to the channel. It's not actually known how ACh activates its ROC, but it's been suggested that it might be through the use of G-proteins (see my post on G-protein coupled receptors here).

Explain the evidence for and differences between electromechanical and pharmacomechanical coupling including the use of extracellular versus intracellular calcium.

Electromechanical coupling seems to imply that a change in voltage, and something to sense the change in voltage (like VOCs), are required in order for contraction to occur. That doesn't happen in all muscles, however, and we know that because of experiments involving nifedipine. Nifedipine blocks L-type Ca²⁺ channels, so if only electromechanical coupling was involved, we'd expect that nifedipine would stop contraction, right?

Wrong.

Nifedipine does have an effect, but only a relatively modest effect. That means that something else must be coming into play here, and that something is pharmacomechanical coupling. Pharmacomechanical coupling occurs via G-protein coupled receptors rather than through ion channels.

Another thing that can be taken away from these experiments is that extracellular Ca²⁺ is not the be-all and end-all. If it was, then blocking Ca²⁺ channels would block contraction. Hence, intracellular Ca²⁺ stores are also important, and I'll get to them in a bit.

Explain how agonist including ACh and NA produce contraction using pharmacomechanical coupling, listing all enzymes, receptors, channels and second messages.

ACh and NA (noradrenaline) can bind to several different receptors. ACh can bind to muscarinic receptors, imaginatively named M₁, M₂, M₃, M₄ and M₅. NA can bind to α-adrenergic and β-adrenergic receptors.

Just to make things a bit confusing, these receptors have different effects. Odd-numbered muscarinic receptors and α-adrenergic receptors can activate a G_q protein, producing inositol triphosphate (IP₃). This causes contraction. Even-numbered muscarinic receptors can activate a G_i protein, inhibiting cAMP (which I assume results in the prevention of relaxation). β-adrenergic receptors activate G_s, activating cAMP and causing relaxation. (For more details on G-protein coupled receptors, please see here.)

When acetylcholine binds to an odd-numbered muscarinic receptor, or noradrenaline binds to an α-adrenergic receptor, G_q activates phospholipase C. Phospholipase C cleaves phosphatidylinositol into inositol triphosphate (IP₃) and diacylglycerol (DAG). For now, we only need to worry about IP₃ as it can bind to IP₃-receptors in the sarcoplasmic reticulum (SR) membrane (remember, the sarcoplasmic reticulum is basically just the endoplasmic reticulum of the muscle cell). These receptors are also Ca²⁺ ion channels, so when they are activated, they release Ca²⁺ stored in the SR. Note that none of this requires a change in membrane potential.

Describe the mechanisms for calcium entry into cells, release from the SR, clearance from cytoplasm, entry into SR and storage in smooth muscle.

As I just mentioned, odd-numbered muscarinic receptors and α-adrenergic receptors can cause release of Ca²⁺ from the SR via IP₃-activated channels. Some smooth muscle SRs also have ryanodine receptors (RyR), which, just like in the heart muscle, produce calcium-induced calcium release. Also, fun fact: RyR receptors can also be activated by caffeine. Ca²⁺ is later pumped back into the SR via the SERCA pump, again like in the heart muscle.

Smooth muscle, as I've alluded to, can also take up extracellular calcium via VOCs and ROCs. Calcium can also be pumped back into the extracellular fluid via Plasma Membrane Calcium ATPase (PMCA) pumps. There are also Na⁺/Ca²⁺ exchangers that can let calcium out in exchange for sodium.

There is also a mechanism for letting in more calcium when intracellular stores are low. STIM is a long transmembrane protein that runs from the inside of the SR, through the SR membrane and almost all the way to the plasma membrane of the cell. It is a Ca²⁺ binding protein which is bound and inactive whenever Ca²⁺ stores are adequate. When Ca²⁺ stores are depleted, it activates SOCCs (store-operated calcium channels) in the plasma membrane that allow entry of more Ca²⁺. SOCCs are conveniently located close to SERCAs so that the calcium can get its way into the SR quickly.

Explain how NA and NO produce relaxation including all enzymes, receptors, channels and second messages.

NA and NO (nitric oxide) have slightly different pathways. Noradrenaline activates adenylate cyclase via a G_s protein. Adenylate cyclase converts ATP to cAMP (cyclic AMP), which activates protein kinase A (PKA). NO, on the other hand, diffuses through the cell membrane and activates guanylate cyclase, which converts GTP to cGMP. cGMP activates protein kinase G (PKG).

PKA and PKG can phosphorylate and open K⁺ channels in smooth muscle, causing hyperpolarisation. They can also increase Ca²⁺ uptake into the SR and reduce Ca²⁺ release from the SR, though the mechanisms here are unknown. (Please note: all of this is completely different to the actions of PKA in cardiac muscle!) PKG can also activate PMCA, which, as I said earlier, pumps Ca²⁺ out of the cell. Since Ca²⁺ causes contraction, getting rid of it causes relaxation.