Saturation curve for radioligand binding assay - (Jun/04/2009 )

Hi!

We're developing some saturation binding curves for a muscarinic cholinergic receptor binding assay. The specific binding curve initially looks as one would expect (i.e. very sharp increase followed by a plateau) but then it all of a sudden takes off at higher radioligand concentrations and keeps increasing.

The non specific binding is low (under 20% of total) and is linear across all the radioligand concentrations.

I was hoping someone might be able to explain why this is happening!

Thanks!

I think you have two binding sites, a low affinity and a high affinity. They probably have different capacities as well. This is fairly common.

That you haven't saturated the high affinity (no plateau) may be because your top concentration is lower than that required for maximal binidng. What did the total binding look like? And how did you block the non-specific sites? What ligand are you using and how specific is it? What do you get when you use established muscarinic ligands specific for each receptor sub-class? Does the increase in bidning occur at the same time point each time?

There was a similar effect reported in the ninties with salmeterol not seen with salbutamol (I think). Salmeterol bound to "holding sites" right nextto the functional binding site. This meant salmeterol had a much longer lasting effect than salbutamol not, as first thought, because of higher affinity but bacause of closer proximity and protection from degredation by being bound on these non-functional sites. Presumably, as the functional binding sites gave up ligand, so the non-functional sites gave up ligand which was then free to bind to functional sites, thereby prolonging the effect.

Interesting piece of pharmacology, isn't it?

Any suggestions for ways to reduce background signal when doing these assays with plate-bound cells?