Probing Molecular and Genetic Mechanisms of Invertebrate Sugar Detection

PROJECT SUMMARY/ABSTRACT Gustatory Receptors (GRs) are a large family of ligand-gated ion channels important for invertebrate chemosensation. While the functional importance of GRs in invertebrate chemosensation is well-established, little has been known about GR structure and function, including how GRs recognize ligands and how ligand binding impacts GR activity. This proposal addresses the mechanisms of GR action by focusing on the Gr43a family of sugar receptors, combining high-resolution structural determination with mutational analysis, ion channel physiology, and molecular genetics and behavior in Drosophila. Aim 1) Probe the mechanisms that control ligand sensitivity and specificity in the Gr43a family: Structure-guided mutational and functional analysis will be used to probe mechanisms of ligand recognition and channel activation. In addition, as Gr43a has multiple alternative isoforms in dipterans, their impact on ligand sensitivity and specificity will be examined. Aim 2) Assess how alternative isoforms of D. melanogaster Gr43a impact its function: Gr43a performs multiple sensory functions in vivo, acting both as a peripheral gustatory receptor and an internal nutrient sensor. Using transgenic and genome edited Drosophila, the hypothesis that different Gr43a isoforms serve distinct in vivo functions will be tested. Aim 3) Examine the impact of activity-altering mutations and isoform variation using cryo-EM: High-resolution cryo-EM-based structures and conformational ensembles will be determined to examine how alternative isoforms of wild type Gr43a and select mutant Gr43a family members impact ligand and channel opening at a mechanistic level. This basic science investigation will provide a detailed understanding of the structural and functional aspects of ligand-binding and channel activation within the large and, to date, poorly understood GR family. Understanding GR activation by ligands is of potential value for disease vector control. GRs play important roles in sensory-driven behaviors in arthropod disease vectors, and GR-expressing neurons are often accessible to environmental chemicals, making GRs promising targets for control agents.