Identification of multiple functional receptors for tyramine on an insect secretory epithelium

Abstract

The biogenic amine tyramine (TA) regulates many aspects of invertebrate physiology and development. Although three TA receptor subtypes have been identified (TAR1-3), specific receptors have not been linked to physiological responses in native tissue. In the Malpighian (renal) tubule of Drosophila melanogaster, TA activates a transepithelial chloride conductance, resulting in diuresis and depolarization of the transepithelial potential. In the current work, mutation or RNAi-mediated knockdown in the stellate cells of the tubule of TAR2 (tyrR, CG7431) resulted in a dramatic reduction, but not elimination, of the TA-mediated depolarization. Mutation or knockdown of TAR3 (tyrRII, CG16766) had no effect. However, deletion of both genes, or knockdown of TAR3 on a TAR2 mutant background, eliminated the TA responses. Thus while TAR2 is responsible for the majority of the TA sensitivity of the tubule, TAR3 also contributes to the response. Knockdown or mutation of TAR2 also eliminated the response of tubules to the related amine octopamine (OA), indicating that OA can activate TAR2. This finding contrasts to reports that heterologously expressed TAR2 is highly selective for TA over OA. This is the first report of TA receptor function in a native tissue and indicates unexpected complexity in the physiology of the Malpighian tubule.

Figure 1

Genomic organization of the TAR2 and TAR3 genes. Boxes indicate exons, with lighter shading indicating coding regions and darker shading indicating untranslated regions. The precise sizes of the deletions created in this study are 12,084 bp (Df(3R)TARΔ124), 10,367 bp (Df(3R)TARΔ30), and 9,294 bp (TAR3 ^Δ29).

Figure 2

(A) Representative responses to 10, 100, and 1000 nM TA of a TAR2 ^f05682 heterozygote (upper trace) and homozygote (lower trace). (B) Dose-response curve for TA in TAR2 ^f05682 heterozygotes and homozygotes. ***Significant difference between genotypes, p < 0.001, 2-way ANOVA and Bonferroni post-test, n = 8–10 tubules per genotype. (C) Response of TAR2 ^f05682 heterozygotes and homozygotes to 100 μM OA. The average response amplitude of homozygotes does not differ from zero, 1-sample t-test, p = 0.76. n = 9 tubules per genotype. (D) Effect of TAR2 and TAR3 RNAi on responses to 100 nM TA (upper graph) and 100 μM OA (lower graph). The responses of RNAi tubules were compared to those of the appropriate parental line by Kruskal-Wallis and Dunn’s multiple comparison tests. Asterices indicate that the only difference seen was upon RNAi of TAR2 in the stellate cells. Knockdown of TAR2 in the stellate cells did not eliminate the response to TA (1-sample t-test, p = 0.01) but did eliminate the response to OA (p = 0.82). n = 8–11 tubules per condition.

Figure 3

Response of TAR deletion mutants to TA. (A) Representative traces showing the response to 1000 nM TA of a Df(3R)TARΔ30 heterozygote (upper trace) and homozygote (lower trace). (B) Dose-response curve to TA for TAR3 ^Δ29, Df(3R)TARΔ30, and Df(3R)TARΔ124 heteroyzygotes and homozygotes. The mean responses of Df(3R)TARΔ30, and Df(3R)TARΔ124 homozygotes to each concentration of TA were either not significantly different from zero by a 1-sample test or were negative. There were no differences in the responses of TAR3 ^Δ29 homozygotes vs heterozygotes, p > 0.05 by 2-way ANOVA and Bonferroni post-test. n = 7–12 tubules per point.

Figure 4

Effect of RNAi against TAR2 and TAR3 on a TAR2 mutant background. (A) Representative responses to 1000 nM TA from c42-gal4 TAR2 ^f05682 /TAR2 ^JF01878 TAR2 ^f05682 (upper trace) and c710-gal4 TAR2 ^f05682 /TAR2 ^JF01878 TAR2 ^f05682 (lower trace) tubules. (B) Representative responses to 1000 nM TA from c42-gal4 TAR2 ^f05682 /TAR3 ^JF02749 TAR2 ^f05682 (upper trace) and c710-gal4 TAR2 ^f05682 /TAR3 ^JF02749 TAR2 ^f05682 (lower trace) tubules. (C) Mean responses to 1000 nM of the four genotypes shown in (A and B). Knockdown of both TAR2 and TAR3 in the stellate cells caused a significant reduction in the response compared to knockdown in principal cells, p < 0.05, 1-way ANOVA and Sidak’s multiple comparisons test, but only knockdown of TAR3 in the stellate cells resulted in a mean response that was equal to zero, p = 0.45, 1-sample t-test. n = 8–9 tubules per genotype.