Conserved Modules Required for Drosophila TRP Function in Vivo

Abstract

Transient receptor potential (TRP) channels are broadly required in animals for sensory physiology. To provide insights into regulatory mechanisms, the structures of many TRPs have been solved. This has led to new models, some of which have been tested in vitro Here, using the classical TRP required for Drosophila visual transduction, we uncovered structural requirements for channel function in photoreceptor cells. Using a combination of molecular genetics, field recordings, protein expression analysis, and molecular modeling, we interrogated roles for the S4-S5 linker and the TRP domain, and revealed mutations in the S4-S5 linker that impair channel opening or closing. We also uncovered differential requirements for the two highly conserved motifs in the TRP domain for activation and protein stability. By performing genetic complementation, we found an intrasubunit interaction between the S4-S5 linker and the S5 segment that contributes to activation. This analysis highlights key structural requirements for TRP channel opening, closing, folding, and for intrasubunit interactions in a native context-Drosophila photoreceptor cells.SIGNIFICANCE STATEMENT The importance of TRP channels for sensory biology and human health has motivated tremendous effort in trying to understand the roles of the structural motifs essential for their activation, inactivation, and protein folding. In the current work, we have exploited the unique advantages of the Drosophila visual system to reveal mechanistic insights into TRP channel function in a native system-photoreceptor cells. Using a combination of electrophysiology (field recordings), cell biology, and molecular modeling, we have revealed roles of key motifs for activation, inactivation and protein folding of TRP in vivo.

Keywords: Drosophila; TRP channels; TRPC; electroretinogram; phototransduction; vision.

Figure 1.

Alanine mutagenesis of the S4–S5 linker, S5px, and the TRP domain. A, Diagram depicting the six transmembrane segments (S1–S6); the S4–S5 linker; the S5-Pore-S6 domain (S5-P-S6); the S5px region, which is the S5 section that is proximal to the S4–S5 linker; and the TRP domain. B, Model of the structure of four Drosophila TRP subunits based on the mouse TRPC4 structure (Duan et al., 2018). The S5-P-S6 domain in each of the four subunits is indicated in (1) red, (2) magenta, (3) blue, and (4) white. C, Ribbon diagram showing key domains in a single TRP subunit. The structure is based on molecular modeling using the structure of mouse TRPC4 as the template (Duan et al., 2018). D, E, Alignment of the amino acid sequences of the S4–S5 linker, S5px, and the TRP domain in the Drosophila TRPCs, human TRPC1, 3–7 and mouse TRPC2. Mouse TRPC2 is shown as human TRPC2 is a pseudogene (Wes et al., 1995). Colors indicate different degrees of conservation: blue, 100% identical; magenta, 80–90% identical; cyan, 50–70% identical; black, <50% identical. The numbers above the sequences correspond to residues in TRP. Residues mutated by alanine substitutions are indicated with green lines above. F, Sixteen residues subjected to alanine (A) mutagenesis. The wild-type amino acid is indicated to the left of the residue number.

Figure 2.

TRP expression levels and modeling of the region including PFN in TRP box 2. A, Western blot showing expression of TRP containing mutations in the S4–S5 linker and S5px. Head extracts were fractionated by SDS-PAGE and probed with rabbit anti-TRP and mouse anti-Tubulin (Tub). trp⁺ is a wild-type trp transgene (sfGFP::TRP). All transgenes were in trp³⁴³ background. Protein size markers are indicated on the left (kDa). B, Western blot showing expression of TRP containing mutations in TRP box 1 and the linker between TRP boxes 1 and 2. C, D, Quantification showing the relative TRP levels detected in the Western blots in A and B. The gray dash line indicates 100% expression, and the green dash line indicates 50% expression. Green labels indicate TRP isoforms expressed at approximately ≥50% expression, and red labels indicate expression of approximately ≤10%, whereas blue indicates expression of ∼25%. E, Western blot showing expression of TRP containing mutations in the TRP box 2. F, Quantification showing the relative TRP levels detected in the Western blots in E. Green label indicates TRP isoform expressed at approximately ≥50% expression, and red labels indicate expression of approximately ≤10%. G, Ribbon diagram indicating the positions and side chains of residue P697, F698, and N699. n = 3. Means ± SEM.

Figure 3.

Effects of trp mutations on the light response using ERGs. A–S, Shown are representative ERG traces from the indicated genotypes. Flies were stimulated with a 10 s pulse of light indicated below each trace. A, Control (w¹¹¹⁸); B, trp³⁴³; C, trp⁺ transgene; D, trp^G533A; E, trp^P534A; F, trp^S538A; G, trp^R541A; H, trp^D545A; I, trp^E675A; J, trp^W676A; K, trp^K677A; L, trp^F678A; M, trp^R680A; N, trp^L683A; O, trp^F688A; P, trp^P695A; Q, trp^P697A; R, trp^F698A; S, trp^N699A. A 10 mV and 10 s time marker is shown. T, Time required for 60% return to the baseline (t₆₀). Comparisons are to trp³⁴³. U, Sustained ERG depolarization (mV) at the end of the 10 s light pulse stimulus. V, Time required for 50% return of the ERG response after cessation of the light stimulus (t₅₀). W, Ribbon diagram showing the hydrogen bond (indicated by the arrow) between R541 in the S5px and E675 in TRP box 1. n ≥ 6. Means ± SEM. One-way ANOVA with Holm-Sidak post hoc analyses. **, p < 0.01.

Figure 4.

Spatial localization of mutated TRP channels in photoreceptor cells. A–I, All of the TRP isoforms were fused to GFP and introduced into a trp³⁴³ mutant background. Whole mounts of compound eyes were stained with anti-GFP (green, top rows) and anti-Rh1 (magenta, middle rows), and optical cross-sections of individual ommatidia are shown at the distal regions, which include the R1-6 and R7 cells. Wild type GFP::TRP is localized to the rhabdomeres of all photoreceptor cells, whereas Rh1 is localized to the rhabdomeres of the R1-6 cells. The merge of the anti-GFP and anti-Rh1 staining is shown in the bottom rows. The compound eyes were dissected and stained from the indicated flies. A, Wild-type trp⁺ transgene (sfGFP::TRP). Scale bar, 5 µm for all panels. B–I, B, trp^P534A; C, trp^S538A; D, trp^R541A; E, trp^E675A; F, trp^K677A; G, trp^R680A; H, trp^L683A; I, trp^F688A. J, Ribbon diagram indicating the side chains and the hydrogen bond (indicated by the arrow) between residues R680, which is localized to the TRP domain, and Q311, which is in the helix-loop-helix.

Figure 5.

Testing trp mutants for dominant ERG phenotypes. A, Flies contained two copies of wild-type transgene (trp⁺) in a trp³⁴³ background. B–H, All flies contained one copy of the indicated transgenes with alanine substitutions and one copy of wild-type transgene (trp⁺) in a trp³⁴³ background. B, trp^P534A; C, trp^S538A; D, trp^R541A; E, trp^E675A; F, trp^K677A; G, trp^L683A; H, trp^F688A. Flies were stimulated with a 10 s pulse of light indicated below each trace. I, Time required for the trp^R541A/trp⁺ ERG to display a 60% return to the baseline (t₆₀). J, Amplitude of sustained ERG responses at the end of the 10 s light stimulus. K, Time required for 50% return of the ERG response after cessation of the light stimulus (t₅₀). n ≥ 6. Means ± SEM.

Figure 6.

Suppression of trp ERG phenotypes in trans-heterozygous flies. Top row and left column indicate the two corresponding mutations analyzed in the trans-heterozygous combinations. Flies were stimulated with a 10 s pulse of light indicated below each trace. The ERG traces are shown in the corresponding boxes.

Figure 7.

Quantification of ERGs of trans-heterozygous flies, shown in Figure 6. A, Quantification of the time required for 60% return to the baseline (t₆₀) of the indicated trans-heterozygous combinations. Comparisons are to trp³⁴³. B, Amplitude of sustained depolarization responses at the end of the 10 s light stimulus. C, Time required for 50% return of the ERG response after cessation of the light stimulus (t₅₀). n ≥ 6. Means ± SEM. D, The backbones and side chains of L535 to R541 showing local hydrogen bonds between these residues, indicated by arrows.