Importance of method validation: Implications of non-correlated observables in sweet taste receptor studies

Running Title: Non-correlation of observables in calcium flux assay of sweet taste receptors ! #! !"#$%&'$! ! Validation of research methodology is critical in research design. Correlation between experimental observables must be established before undertaking extensive experiments or propose mechanisms. This article shows that, observables in the popular calcium flux strength assay used in the characterization of sweetener-sweet taste receptor (STR) interaction are uncorrelated. In pursuit to find potential sweeteners and enhancers, calcium flux generated via G-protein coupling for wildtype and mutant receptors expressed on cell surface is measured to identify and localize sweetener binding sites. Results are channeled for sweetener development with direct impact on public health. We show that flux strength is independent of EC50 and sweet potency. Sweet potency-EC50 relation is non-linear and anti-correlated. Single point mutants affecting receptor efficiency, without significant shift in EC50 have been published, indicating flux strength is independent of ligand binding. G-protein coupling step is likely observed in the assay. Thus, years have been spent generating uncorrelated data. Data from uncorrelated observables does not give meaningful results. Still, majority of research in the field, uses change in calcium flux strength to study the receptor. Methodology away from flux strength monitor is required for sweetener development, reestablish binding localization of sweeteners established by flux strength method. This article serves to remind researchers to validate methodology before plunging into long term projects. Ignoring validation test on methodology, have been a costly mistake in the field. Concepts discussed here is applicable, whenever observable in biological systems are many steps moved from the event of interest.! ! $! Introduction The sweet taste receptor, binds to small molecule sweeteners and large molecular weight sweet tasting proteins, and elicits sweet taste. It is an important research target, in sweetener development, synergy studies to reduce total amount of sweetener in foods and for health benefits to reduce obesity and diabetes. Tas I genes encode protein sequences for T1R2-T1R3 sweet taste receptor and T1R1-T1R3 umami taste receptor [1-5]. Umami taste receptor is responsible for taste of MSG. Many present working hypotheses in the field is derived from concepts in mGluR [6-10], the best characterized receptor of Class C GPCR. Publications in the field popularly use G-protein generated cell based activity assay to monitor receptor-ligand binding. Experiments involve co-transfection of pairs of wildtype, mutant or chimeric GPCR cDNAs into heterologous cell lines. Activity of the receptor expressed in eukaryotic cells, is …