Dysregulation of nicotinic acetylcholine receptors (nAChRs) is implicated in a wide variety of disorders, including nicotine addiction and Alzheimer’s disease. However, it has been challenging to target nAChRs pharmacologically in disease states because the receptors are comprised of structurally similar subunits that are found broadly throughout the nervous system. Another challenge is avoiding excessive activation of these receptors, which can cause cytotoxicity. One way to circumvent these challenges could be to identify and target different proteins that regulate and modulate nAChRs. In this thesis, we describe two such proteins, Ly6h in mammals and Sleepless in flies; we describe the mechanisms by which these Ly6 proteins regulate nAChRs; and we identify structural motifs that enable these regulatory functions. Specifically, using ion flux assays, co-immunoprecipitations, surface labeling, and confocal microscopy, we show that both Ly6h and Sleepless utilize a single loop to interact with and inhibit nAChRs, at least in part by preventing accumulation of functional receptors on the cell surface. Additionally, in mammalian cells we identify direct antagonism between Ly6h and NACHO, a recently discovered chaperone that can accelerate the assembly of nAChRs. We demonstrate that this antagonism between Ly6h and NACHO is likely to result from competition for binding and assembly of nAChRs. These studies provide insight into mechanisms through which nAChRs are likely to be tightly regulated, which may help in the design of novel drugs to modulate cholinergic signaling in disease states.