Figure 6: Whole-cell voltage clamp recordings of S2 cells expressing GRs.

Cells were stimulated with 30 mM L-canavanine as indicated. (a,b) Cells were transfected either with pActin5c–GAL4, pUAST–EGFP only (mock) or pActin5c–GAL4, pUAST–EGFP plus pUAST–Gr8a, pUAST–Gr66a and pUAST–Gr98b (3 GRs). (a) Currents produced in response to voltage steps (−80 mV to +80 mV in 20 mV increments) of 500-ms duration obtained in the presence of L-canavanine. (b) I–V relationships using cells expressing GR8a, GR66a and GR98b, and stimulated with L-canavanine. (c) Current densities at +80 mV. The cells expressed the indicated GRs and were recorded in the presence or absence of L-canavanine stimulation. The numbers of recordings are indicated. *P<0.05 (paired Student’s t-test). (d-f) Current–voltage traces showing that expression of two GRs in S2 cells did not lead to L-canavanine-induced increases in current densities. (d) Gr8a- and Gr66a-expressing S2 cells (n=10). (e) Gr8a- and Gr98b-expressing cells (n=11). (f) Gr66a- and Gr98b-expressing cells (n=6). (g) Effect of La³⁺ on I–V relationship. The cells expressed GR8a, GR66a and GR98b, and were recorded in the presence of L-canavanine. (h) Effect of La³⁺ on current densities obtained at +80 mV. The cells expressed the three GRs and were stimulated with L-canavanine. **P<0.01 (analysis of variance (ANOVA) with post hoc Tukey test). (i) Current densities in cells expressing GR8a, GR66a and GR98b and stimulated with the indicated bitter chemicals: 30 mM L-canavanine, 1 mM papaverine (PAP), 1 mM strychnine (STR), 1 mM denatonium (DEN), 100 μM berberine (BER), 1 mM lobeline (LOB), 5 mM caffeine (CAF), and 1 mM quinine (QUIN). The numbers of recordings are indicated. **P<0.01 (ANOVA with post hoc Tukey test). All error bars indicate s.e.m.