The uses and abuses of molarity — II

Just as the last post showed why a 1 Molar solution of a protein makes no sense at all, it is reasonable to ask what the highest concentration of a single protein in the cellular environment could be. Strangely, it was very hard for me to find an estimate of the percentage of protein mass inside a eukaryotic cell. There is one for the red blood cell, which is essentially a bag of hemoglobin. The amount is 33 grams/deciliter or 330 grams/liter. Hemoglobin (which is a tetramer) has a molecular mass of 64,000 Daltons.  So that’s 330/64000 = .5 x 10^-3 Molar.   So all proteins in our cells have a maximum concentration at most in the milliMolar range.

Before moving on, how do you think the red blood cell gets its energy?  Amazingly it is by anaerobic glycolysis, not using the oxygen carried by hemoglobin at all.  Why? If it used oxidative phosphorylation which runs on oxygen, it would burn up.  That’s why red cells do not contain mitochondria. 

On to Kd the dissociation constant.  At least 475 FDA approved drugs target G Protein Coupled Receptors (GPCRs), and our genome codes for some 826 of them.  Almost 500 of them code for smell receptors, and of the 300 or so not involved with smell 1/3 are orphans (as of 2019) with no known ligand.  There are GPCRs for all neurotransmitters which is why neurologists and psychiatrists are very interested in them. 

The Kd is defined as [ free ligand ][ free receptor ]/ [ ligand bound to receptor]  where all the  [  ]’s are concentrations in Moles/liter (e.g. Molar concentrations). 

There’s the rub.  Kd makes sense when ligand and receptor are swimming around in solution, but GPCRs never do this.  The working GPCR is embedded in our cell membrane which topologists tell us are 2 dimensional manifolds embedded in 3 dimensional space.  What does concentration mean in a situation like this?  Think of the entropy involved in getting all the GPCRs to lie in a single plane.  Obviously not so simple.  

People get around this by using radioactive ligands, and embedding GPCRs in membranes and measuring the time for ligands to bind and unbind (e.g. kinetics), but this is miles away from the physiologic situations — for details please see

Mol Cell Endocrinol. 2019 Apr 5; 485: 9–19.

doi: 10.1016/j.mce.2019.01.018

 

The same is true for other proteins of interest — ion channels for the neurologist, hormone receptors for the endocrinologist, angiotension converting enzyme 2 (ACE2) for the pandemic virus.  

 

I think that all Kd’s of membrane embedded receptors do is give you an ordinal ordering (e.g. receptor A binds ligand B tighter than ligand C ) but not a quantitative one.

 

Next up, how a Nobel prizewinner totally misunderstood the nature and applicability of molarity and studies on a two dimensional gas (complete with Pressure * Area = n * Gas Constant * Temperature).

 

 

 

Do orphan G Protein Coupled Receptors self stimulate?

Self-stimulation is frowned on in the Bible — Genesis 38:8-10, but one important G Protein Coupled Receptor (GPCR) may actually do it.  At least 1/3 of the drugs in clinical use manipulate GPCRs, and we have lots of them (at least 826/20,000 protein coding genes according to PNAS 115 p. 12733 ’18).  However only 360 or so are not involved in smell, and in one third of them  we have no idea what the natural ligand for them actually is (Cell vol. 177 p. 1933 ’19).  These are the orphan GPCRs, and they make a juicy target for drug discovery (if only  we knew what they did)

One orphan GPCR goes by the name of GPR52. It is found on neurons carrying the D2 dopamine receptor.  GPR52 binds to G(s) family of G proteins stimulating the production of CAMP (which would antagonize dopamine signaling), enough to stimulate (if not self-stimulate) any neuropharmacologist.

Which brings us to the peculiar behavior of GPR52 as shown by Nature vol. 579 pp. 142 – 147 ’20.  The second extracellular loop (ECL2) folds into what would normally be the binding site for an exogenous ligand (the orthosteric site).  Well, it could be protecting the site from inappropriate ligands.  But it isn’t, as removing or mutating ECL2 decreases the activity of GPR52 (e.g. less CAMP is produced).  Pharmacologists have produced a synthetic GPR52 agonist (called c17).  However it binds to a side pocket, in the 7 transmembrane region of the GCPR.   This is interesting in itself, as no such site is known in any of the other GPCRs studied.

Most GPCRs have some basal (constitutive) activity where they spontaneously couple to their G proteins, but the constitutive activity of GPR52 is quite high, so c17 only slightly increases the rise in CAMP that GPR52 normally produces.

This might be an explanation for other orphan GPCRs — like a hermaphrodite they could be self-fertilizing.