3.3. Orco functions like a TRP channel in P. pastoris
VUAA1 is one of the most potent agonists of Orco and was once a prime
candidate for use as a repellant since it was reported to overstimulate
mosquitoes to the point of confusion [32]. Unfortunately, VUAA1 also
exhibits exceptionally low volatility, which presents significant
challenges to its use as a repellant. Nevertheless, it is an excellent
ligand to test the behavior of the Pichia biosensor in liquid
cultures. We exposed the PP-Orco cells to increasing concentrations of
VUAA1 in the presence of 1 mM (Figure 4 ) and 5 mM of
Ca2+ (Figure 5 ) in Hank’s buffer. VUAA1 was
added to the PP-Orco cultures by suitably diluting a stock solution of
the molecule in DMSO. In the case of PP-Orco cultures that are exposed
to 1 mM of extracellular Ca2+, exposure to 0.125 mM
and 0.25 mM of VUAA1 did not produce a statistically significant
response compared to unmodified P. pastoris GS115 cells but
concentrations over 0.50 mM generate a distinct, statistically
significant response compared to the control. The p-values for the
difference in normalized fluorescence between PP-Orco and GS115 strains
were lower than 0.01 for the 0.5 mM, 1 mM and 2 mM runs and 0.001 when
the concentration of VUAA1 was 1.5 mM.