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Fig. 1. Responses of Oocytes to Sodium Chloride and Sodium Bicarbonate
Representative traces obtained with the following types of oocytes from the same batch and age:
(A) Intact (naked) Xenopus oocytes.
(B) Oocyte coexpressing CquiGR1 and CquiGR3
(C) Oocyte coexpressing CquiGR1 and CquiGR2
(D) Oocyte coexpressing CquiGR2 and CquiGR3.
All scales are 200 nA and 0.5 min. Oocytes were first challenged with 200 mM NaCl and then 200 mM NaHCO3.
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Fig. 2. Responses of CquiGR2+CquiGR3-coexpressing oocytes to sodium chloride and sodium bicarbonate
(A) Representative trace from a single oocyte preparation stimulated with increasing doses (10 – 300 mM) of the two compounds. For clarity, responses to sodium chloride and sodium bicarbonate were colored red and blue, respectively.
(B) Concentration-dependent curves obtained with five different oocytes coexpressing CquiGR2 and CquiGR3.
Data are presented as mean ± SEM. Some error bars for NaCl curve do not appear, because they are shorter than the size of the symbol. From left to right: 0.81, 2.59, 3.24, 5.35, 6.92, 11.24, 9.59, and 14.42.
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Fig. 3. Comparative concentration-dependent curves for oocytes coexpressing either two or three functional subunits
Results were obtained with four oocytes coexpressing CquiGR2 and CquiGR3 and four oocytes coexpressing the three GRs. For simplicity, in figures, we use two-letter symbols for proteins as opposed to four-letter symbols (eg, Cq = Cqui) used elsewhere. Data are presented as mean ± SEM. Error bars for the green curve do not appear because they are shorter than the size of the symbol. From left to right: 0, 0.63, 3.24, 1.65, and 3.84.
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Fig. 4. Effect of different variants of CquiGR1 on the response of CquiGR2+CquiGR3-coexpressing oocytes
All experiments were performed with four replicates with oocytes from the same batch coexpressing either CquiGR2+CquiGR3 or CquiGR2+CquiGR3 plus one of the CquiGR1 variants (K456N, M365V, D143G;R434K, and L246F). The clone with sequence identical to that in VectorBase (CJPI 006622) was named wild type (wt). Responses recorded from CquiGR2+CquiGR3-coexpressing oocytes and those recorded from oocytes coexpressing CquiGR2+CquiGR3 plus one CquiGR1variant after stimulus with 200 mM sodium bicarbonate (n = 4) were compared by unpaired t-test. P values are presented on the top of each variant column. Data are presented as mean ± SEM.
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Fig. 5. CO2 concentrations in solutions of sodium bicarbonate prepared in Ringer buffer
The calculated concentrations based on the final pH of each preparation (n = 4) are shown in red. The actual concentration of CO2 in each solution was measured with a CO2 Ion Selective Electrode. Data are presented as mean ± SEM.
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Fig. 6. Effect of pH on the oocyte response and the concentrations of CO2 in solution (A) Responses of CquiGR2+CquiGR3-expressing oocytes to 10 mM of sodium bicarbonate without acidification and after acidification (n = 12). The final pH (pHlowered) of the sodium bicarbonate buffer was 7.39 ± 0.05. (B) Quantification of CO2 in sodium bicarbonate samples (n = 4) without and after pH adjustment (acidification) as measured with a CO2 Ion Selective Electrode. After acidification (pHlowered), the pH of the sodium bicarbonate samples was 7.32 ± 0.03. Data are presented as mean ± SEM.
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Fig. 7. Responses of An. gambiae GRs-expressing oocytes to sodium chloride and sodium bicarbonate
(A) Trace obtained with an oocyte coexpressing AgamGR23 and AgamGR24. (B) Responses of another oocyte (from the same batch) co-expressing the three GRs from An. gambiae, GR22, GR23, and GR24. (C) Replication of experiment in (A), but using a different batch of oocytes. (D) Replication of the experiment in (B) with the same batch of oocytes used in (C). Δ values represent responses to sodium bicarbonate subtracted from responses to sodium chloride at the same concentration. In a third replication with a different batch of oocytes the Δ values recorded from a GR23+GR24-coexpressing oocyte stimulated with 50, 100, 200, and 300 mM were 120, 225, 250, and 780 nA, whereas the respective values recorded from an oocyte from the same batch and coexpressing GR22+GR23+GR24 were 23, 89, 193, and 270 nA.
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S1. NaCl and NaHCO3 responses recorded from oocytes only and different ooocytes coexpressing combination of Culex GRs
From top to bottom: naked oocytes (black), oocytes coexpressing GR1+GR3 (red), GR1/GR2-coexpressing oocytes (green), and oocytes coexpressing GR2 and GR3. NaCl and NaHCO3 were applied at 200 mM.
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S2. Effect of CquiGR1 variants on the modulation of CO2 response
Traces obtained with the same batch of oocytes injected either with CquiGR2+CquiGR3 or a ternary combination of CquiGR2+CquiGR3 plus one of the CquiGR1 variants. For clarity, responses to sodium chloride and sodium bicarbonate are colored red and blue, respectively.
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S3. Responses obtained with a CquiGR2+CquiGR3-expressing oocyte when stimulated with carbon dioxide
Samples were prepared by bubbling CO2 in Ringer buffer and adjusting the final pH or by dissolving either sodium chloride or sodium bicarbonate in Ringer buffer. The concentrations of CO2 (in samples prepared by bubbling CO2 or by dissolving NaHCO3) were measured using a CO2 Ion Selective Electrode. Of note, currents elicited by sodium carbonate are larger than expected for the amount of CO2 (aq), because they include currents elicited by sodium.
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S4. Responses of Ae. aegypti GRs coexpressed in Xenopus oocytes and challenged by sodium chloride and sodium bicarbonate
(A) Traces obtained with an AaegGR2/AaegGR3-coexpressing oocyte. (B) Responses recorded from another oocyte of the same age and the same batch, but coexpressing the subunits AaegGR1, AaegGR2, and AaegGR3. Δ values represent responses to sodium bicarbonate subtracted from responses to sodium chloride at the same concentration. 4
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