Control of Synaptic Levels of Nicotinic Acetylcholine Receptor by the Sequestering Subunit Da5 and Secreted Scaffold Protein Hig

The presentation of nicotinic acetylcholine receptors (nAChRs) on synaptic membranes is crucial for generating cholinergic circuits, some of which are associated with memory function and neurodegenerative disorders. Although the physiology and structure of nAChR, a cation channel comprising five subunits, have been extensively studied, little is known about how the receptor levels in interneuronal synapses are determined and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices and intracellular proteins. By a genetic screen of Drosophila, we identified mutations in the nAChR subunit Da5 gene as suppressors that restored the mutant phenotypes of hig, which encodes a secretory matrix protein localized to cholinergic synaptic clefts in the brain. Only the loss of function of Da5 among the 10 nAChR subunits suppressed hig mutant phenotypes in both male and female flies. Da5 behaved as a lethal factor when Hig was defective; loss of Da5 in hig mutants rescued lethality, upregulating Da6 synaptic levels. By contrast, levels of Da5, Da6, and Dal subunits were all reduced in hig mutants. These three subunits have distinct properties for interaction with Hig or trafficking, as confirmed by chimeric subunit experiments. Notably, the chimeric Da5 protein, which has the extracellular sequences that display no positive interaction with Hig, exhibited abnormal distribution and lethality even in the presence of Hig. We propose that the sequestering subunit Da5 functions by reducing synaptic levels of nAChR through internalization, and this process is blocked by Hig, which tethers Da5 to the synaptic cleft matrix.