Mechanisms of Neurotransmitter-gated Ion Channels

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DESCRIPTION (provided by applicant): The muscle nicotinic acetylcholine receptor channel (AChR) is a ligand-gated ion channel that mediates fast synaptic transmission at the vertebrate neuromuscular junction. Other than its obviously relevant role during normal neurotransmission, the AChR has played a pivotal role in our understanding of synaptic ionotropic receptors. This proposal covers facets of the two major functional aspects of ligand-gated ion channels, namely, binding-gating-desensitization and ion permeation. With regard to the triad binding-gating-desensitization, our long-term goal is to understand how these three intertwined phenomena shape synaptic transmission through the muscle nicotinic acetylcholine receptor (AChR). With regard to ion permeation, our long-term goal is to understand the role of the six rings of acidic/basic amino-acid residues that flank the pore domain of the AChR. Although it is clear that they must somehow modulate the concentration of cations at the ends of the pore, the problem is still far from being understood in its full complexity. Experiments are proposed to characterize these aspects of the AChR's function through quantitative analysis of single-channel and macroscopic-current patch-clamp recordings performed on cells that heterologously express recombinant mouse receptors. The first goal is to test the hypothesis that acetylcholine can fall off of the binding sites when the channel is open. This is a fundamental tenet of theories of allosteric protein activation. The second goal is to understand how the ligand affinities for the closed and open states change from agonist to agonist to give rise to a range of efficacies. The third goal is to estimate the extent to which entry into desensitization contributes to neuromuscular synaptic transmission in pathological conditions (slow-channel congenital myasthenic syndromes). The fourth goal is to understand how the pore-flanking rings of acidic/basic residues affect ion conduction, and how their pKa-values are modulated by the channel's protein environment.