Cavities in Choline Acetyltransferase and Neuromuscular Disorders

We propose to determine how congenital missense mutations in choline acetyltransferase (ChAT), which synthesizes the prototypical neurotransmitter acetylcholine, decrease enzyme function to cause a severe neuromuscular disorder known as congenital myasthenic syndrome with episodic apnea (CMS-EA). Our structural studies have shown that known CMS-EA mutations are distributed widely over the enzyme despite their common effects on function, and that at least two of these mutations affect the conformation of the catalytic histidine residue. Further, the core residue packing in ChAT is poor, leaving an unusually large number of cavities (voids). Filling one of these cavities ameliorates the effects of a nearby congenital mutation. Based on these observations, we hypothesize that CMS-EA point mutations cause structural changes that propagate to disrupt ChAT function and that this structural instability results from the large number of core cavities. We will use a combination of structural, biophysical, and cell biological approaches to test this hypothesis and to lay the foundation for a therapeutic approach to treating CMS-EA. Two specific aims are proposed: 1) understand the role of poor core packing in mediating the sensitivity of ChAT to missense point mutations, 2) develop cavity-binding ligands that reduce the effects of one or more congenital mutations. This work will define the mechanistic basis for a molecular disease, explore basic principles of protein core packing, and guide development of a treatment for this neuromuscular disorder.