Engineering the acetylcholine receptor gating conformational change
Jadey, Snehal V.
MetadataShow full item record
Acetylcholine receptors (AChRs) are allosteric proteins that respond to acetylcholine (ACh) in the extracellular solution. These membrane proteins alternate spontaneously (`gate') between two global shapes, R and R*. R has a low affinity for ACh and a closed ion channel, and R* has a high affinity and an open channel. We used single channel electrophysiology along with site directed mutagenesis to study the function of these receptors in response to various different structurally relevant nicotinic agonists on wt and mutant receptors. By solving the long standing technological problem associated with bandwidth limitations and ion channel block by agonists, I have engineered the receptor to work in a predictable fashion and in a range where the rate and equilibrium constants can be accurately estimated. On studying various partial and full agonists we proposed a mechanism which suggested that agonist binding to receptors in not by diffusion alone, whereas it involves the binding sites to undergo two conformational changes "catch" and "hold" that connect the apo-, low affinity bound and the high affinity bound structures. We also proposed that loop C capping which is an important structural feature seen in crystal structures, mainly reflects agonist binding. Further I also conducted extensive work on a residue αG153 with many different agonists including nicotine. This residue is the only known position so far, mutating which increases the affinity for different agonists. All the agonists tested on different αG153 side chains showed a modest increase in affinity towards the resting receptor. However, only Nicotine on lys side chain showed an extraordinary effect on the resting affinity. We speculated that neuronal AChRs which have a lys at the same position, may be exposed to a nicotine-like compound during the normal physiology.