The regulation of energy balance is controlled, in part, by nutrient-dependent stimuli via vagal-afferent signalling, and this can be disrupted in obesity. The ability of cholecystokinin to induce postprandial satiety at the type 1 cholecystokinin receptor (CCK1R) makes this receptor an anti-obesity target. However, CCK1R agonists failed in their primary endpoints in clinical trials with obese patients. Proof-of-concept studies have shown correlation of high membrane cholesterol with aberrant CCK1R stimulus-activity coupling; with increased agonist affinity and paradoxical reduced biological responses. To mimic the high cholesterol state, we used cholesterol loaded methyl-beta-cyclodextrin (MβCD) and a high cholesterol mimetic Y140A mutant. In our hands, the former was highly variable with respect to changes in membrane cholesterol, whereas the latter approach was highly reproducible and allowed us to identify a unique ability of this high cholesterol mimetic to induce G protein trapping in BRET-based G protein rearrangement assays. Due to the presumed reduced stability of the Y140A mutant, its isogenic cell exhibited lower cell-surface receptor expression compared to WT. After receptor depletion using the CCK1R pseudo-irreversible antagonist devazepide, we were able to demonstrate that the G protein trapping at the Y140A mutant did not appear to be a consequence of low surface expression. We have also established a method to isolate, culture and perform calcium imaging on primary nodose ganglia neurons from mice, providing a key tool for future translation. Our studies offer insights into cholesterol sensitivity of CCK1R signalling, which will aid in the development of more effective drugs to treat obesity.