The genetic architecture of degenerin/epithelial sodium channels in Drosophila

Abstract

Degenerin/epithelial sodium channels (DEG/ENaC) represent a large family of animal-specific membrane proteins. Although the physiological functions of most family members are not known, some have been shown to act as nonvoltage gated, amiloride-sensitive sodium channels. The DEG/ENaC family is exceptionally large in genomes of Drosophila species relative to vertebrates and other insects. To elucidate the evolutionary history of the DEG/ENaC family in Drosophila, we took advantage of the genomic and genetic information available for 12 Drosophila species that represent all the major species groups in the Drosophila clade. We have identified 31 family members (termed pickpocket genes) in Drosophila melanogaster, which can be divided into six subfamilies, which are represented in all 12 species. Structure prediction analyses suggested that some subunits evolved unique structural features in the large extracellular domain, possibly supporting mechanosensory functions. This finding is further supported by experimental data that show that both ppk1 and ppk26 are expressed in multidendritic neurons, which can sense mechanical nociceptive stimuli in larvae. We also identified representative genes from five of the six DEG/ENaC subfamilies in a mosquito genome, suggesting that the core DEG/ENaC subfamilies were already present early in the dipteran radiation. Spatial and temporal analyses of expression patterns of the various pickpocket genes indicated that paralogous genes often show very different expression patterns, possibly indicating that gene duplication events have led to new physiological or cellular functions rather than redundancy. In summary, our analyses support a rapid early diversification of the DEG/ENaC family in Diptera followed by physiological and/or cellular specialization. Some members of the family may have diversified to support the physiological functions of a yet unknown class of ligands.

Keywords: Degenerin/epithelial sodium channel; chemosensation; fruit fly; mechanosensation; phylogeny.

Figure 1

(A) Illustration depicting a typical DEG/ENaC subunit. TM, transmembrane domain; Red circles represent conserved cysteines; yellow circle represents the “DEG” residue, which in some subunits results in a constitutively open channel state when mutated (Adams et al. 1998; Kellenberger et al. 2002; Snyder et al. 1998, 2000). (B) The protein sequence of PPK, one of the first DEG/ENaC subunits that was identified in the Drosophila genome (Adams et al. 1998). Alignment of all the Drosophila subunits described in Table 1 and Table S1 indicate the presence of a highly conserved cysteine-enriched domain (also see Figure 7A, thumb domain), highlighted in green. Conserved cysteines are highlighted in red; DEG, a predicted “deg” residue, is highlighted in yellow. TM1 and TM2 represent the predicted transmembrane domains 1 and 2, respectively.

Figure 2

Maximum-likelihood unrooted phylogenetic tree inferred from multiply aligned amino acid sequences for D. melanogaster DEG/ENaC ppk genes. A total of 31 DEG/ENaC amino acid sequences are divided into six clusters and labeled as groups I-VI. Bootstrap values are given on branches and amino acid substitution rate is given at the bottom of the figure. Colors represent chromosomally clustered subunits (see Figure 5 for details).

Figure 3

(A) Spatial expression patterns of ppk genes. Microarray expression data were extracted from FlyAtlas (Chintapalli et al. 2007). Expression represents the average signal from four independent microarrays. (B) Temporal expression patterns of ppk genes. Data were extracted from the modENCODE RNA-seq database (Celniker et al. 2009). Expression levels are represented as log₂ values of the original coverage. Numbers at the tops of truncated bars show actual expression values.

Figure 4

ppk and ppk26 expression in larval multidendritic neurons. (A) ppk-GAL4 x UAS-mCD8::GFP. (B) ppk26-GAL4 x UAS-mCD8::GFP. White arrows indicate cell body. (C) Alignment of ppk, rpk, and ppk26 amino acid sequence. Green, residues are conserved across all proteins examined; yellow, residues are conserved in some species; blue, conserved substitutions.

Figure 5

Chromosomal clusters of ppk genes. (A) Cluster of ppk7 and ppk14 located at 2L: 26C3-26C3. (B) Cluster of ppk18, ppk16, and ppk11 located at 2L: 30C8-30C9. Note that although CG13121 is currently annotated as a separate gene, molecular analyses of mRNA clones indicate that it is part of the ppk18 locus (not shown). (C) Cluster of ppk21, ppk20, ppk30, and ppk19 located at 3R: 99B6-99B7. Black boxes, ppk genes; gray boxes, none-ppk genes.

Figure 6

Structural modeling of the ppk family in Drosophila. (A) Domain organization of the chicken ASIC1a subunit (Jasti et al. 2007) Red: TM1 (left helix), TM2 (right helix); yellow: Palm; cyan: Knuckle; orange: beta-ball; purple: Finger; green: Thumb. (B) ASIC1a subunit rendered by conservation information from its alignment with the ppk family. The regions colored in purple are highly conserved residues, whereas those colored in red are most variable in the alignment. (C) Predicted structure for all Drosophila PPK subunits. The rainbow scale represents the residue conservation scores. The regions colored in red are most variable whereas regions in blue are highly conserved.

Figure 7

(A) The alignment of individual subunits from ppk subfamily Group V (for full Group V alignment, see Figure S1). The dashed frame marks the unstructured loop region. Note that PPK17 does not have the unstructured loop region. Q1XA76 is the chicken ASIC Uniprot Accession ID. Consensus sequence was built from the majority of the aligned residues. The bars in the bottom represent conservation percentage after alignment. (B) Unstructured loop region in the subfamily Group V. Predicted structures for all D. melanogaster PPK subunits are shown in Figure 6. The rainbow scale represents residue conservation as in Figure 6.