Phototransduction in Drosophila Is compromised by Gal4 expression but not by InsP3 receptor knockdown or mutation

Drosophila phototransduction is mediated by phospholipase C, leading to activation of transient receptor potential (TRP) and TRP-like (TRPL) channels by mechanisms that are unresolved. A role for InsP(3) receptors (IP(3)Rs) had been excluded because IP3R mutants (itpr) appeared to have normal light responses; however, this was recently challenged by Kohn et al. ("Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo," Journal of Neuroscience 35: 2530), who reported defects in phototransduction after IP3R-RNAi knockdown. They concluded that InsP3-induced Ca2+ release plays a critical role in facilitating channel activation, and that previous failure to detect IP3R phenotypes resulted from trace Ca2+ in electrodes substituting for InsP(3)-induced Ca2+ release. In an attempt to confirm this, we performed electroretinograms, whole-cell recordings, and GCaMP6f Ca2+ imaging from both IP3R-RNAi flies and itpr-null mutants. Like Kohn et al., we used GMRGal4 to drive expression of UAS-IP3R-RNAi, but we also used controls expressing GMRGal4 alone. We describe several GMRGal4 phenotypes suggestive of compromised development, including reductions in sensitivity, dark noise, potassium currents, and cell size and capacitance, as well as extreme variations in sensitivity between cells. However, we found no effect of IP3R RNAi or mutation on photoreceptor responses or Ca2+ signals, indicating that the IP3R plays little or no role in Drosophila phototransduction

Phototransduction in Drosophila Is Compromised by Gal4 Expression but not by InsP3 Receptor Knockdown or Mutation

$\textit{Drosophila}$ phototransduction is mediated by phospholipase C, leading to activation of transient receptor potential (TRP) and TRP-like (TRPL) channels by mechanisms that are unresolved. A role for InsP$_3$ receptors (IP$_3$Rs) had been excluded because IP$_3$R mutants ($\textit{itpr}$) appeared to have normal light responses; however, this was recently challenged by Kohn et al. ("Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of $\textit{Drosophila}$ photoreceptors in vivo," Journal of Neuroscience 35:2530), who reported defects in phototransduction after IP$_3$R-RNAi knockdown. They concluded that InsP$_3$-induced Ca$^{2+}$ release plays a critical role in facilitating channel activation, and that previous failure to detect IP$_3$R phenotypes resulted from trace Ca$^{2+}$ in electrodes substituting for InsP$_3$-induced Ca$^{2+}$ release. In an attempt to confirm this, we performed electroretinograms, whole-cell recordings, and GCaMP6f Ca$^{2+}$ imaging from both IP$_3$R-RNAi flies and $\textit{itpr}$-null mutants. Like Kohn et al., we used GMRGal4 to drive expression of UAS-IP$_3$R-RNAi, but we also used controls expressing GMRGal4 alone. We describe several GMRGal4 phenotypes suggestive of compromised development, including reductions in sensitivity, dark noise, potassium currents, and cell size and capacitance, as well as extreme variations in sensitivity between cells. However, we found no effect of IP$_3$R RNAi or mutation on photoreceptor responses or Ca$^{2+}$ signals, indicating that the IP$_3$R plays little or no role in $\textit{Drosophila}$ phototransduction.Biotechnology and Biological Sciences Research Council: 501100000268, BB/G0092531/1, BB/M007006/1 (RCH, MKB, C-HL) European Union’s Horizon 2020 (EU Framework Programme for Research and Innovation), 501100007601 (RCH, SA: EU Grant 658818), and the Erasmus program (MEK)

A Single Residue Mutation in the Gαq Subunit of the G Protein Complex Causes Blindness in Drosophila.

Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with previously reported strong loss-of-function mutations remain responsive to light stimuli. We recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional electroretinogram assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least five orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. Our new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector, PLC. Our study has thus uncovered possibly the first point mutation that specifically affects this interaction in vivo

A Systematic Mapping Approach of 16q12.2/FTO and BMI in More Than 20,000 African Americans Narrows in on the Underlying Functional Variation: Results from the Population Architecture using Genomics and Epidemiology (PAGE) Study

Genetic variants in intron 1 of the fat mass- and obesity-associated (FTO) gene have been consistently associated with body mass index (BMI) in Europeans. However, follow-up studies in African Americans (AA) have shown no support for some of the most consistently BMI-associated FTO index single nucleotide polymorphisms (SNPs). This is most likely explained by different race-specific linkage disequilibrium (LD) patterns and lower correlation overall in AA, which provides the opportunity to fine-map this region and narrow in on the functional variant. To comprehensively explore the 16q12.2/FTO locus and to search for second independent signals in the broader region, we fine-mapped a 646-kb region, encompassing the large FTO gene and the flanking gene RPGRIP1L by investigating a total of 3,756 variants (1,529 genotyped and 2,227 imputed variants) in 20,488 AAs across five studies. We observed associations between BMI and variants in the known FTO intron 1 locus: the SNP with the most significant p-value, rs56137030 (8.3×10-6) had not been highlighted in previous studies. While rs56137030was correlated at r2>0.5 with 103 SNPs in Europeans (including the GWAS index SNPs), this number was reduced to 28 SNPs in AA. Among rs56137030 and the 28 correlated SNPs, six were located within candidate intronic regulatory elements, including rs1421085, for which we predicted allele-specific binding affinity for the transcription factor CUX1, which has recently been implicated in the regulation of FTO. We did not find strong evidence for a second independent signal in the broader region. In summary, this large fine-mapping study in AA has substantially reduced the number of common alleles that are likely to be functional candidates of the known FTO locus. Importantly our study demonstrated that comprehensive fine-mapping in AA provides a powerful approach to narrow in on the functional candidate(s) underlying the initial GWAS findings in European populations

A saturated map of common genetic variants associated with human height

Common single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40-50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes(1). Here, using data from a genome-wide association study of 5.4 million individuals of diverse ancestries, we show that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a mean size of around 90 kb, covering about 21% of the genome. The density of independent associations varies across the genome and the regions of increased density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs (or all SNPs in the HapMap 3 panel(2)) account for 40% (45%) of phenotypic variance in populations of European ancestry but only around 10-20% (14-24%) in populations of other ancestries. Effect sizes, associated regions and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely to be explained by linkage disequilibrium and differences in allele frequency within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than are needed to implicate causal genes and variants. Overall, this study provides a comprehensive map of specific genomic regions that contain the vast majority of common height-associated variants. Although this map is saturated for populations of European ancestry, further research is needed to achieve equivalent saturation in other ancestries. A large genome-wide association study of more than 5 million individuals reveals that 12,111 single-nucleotide polymorphisms account for nearly all the heritability of height attributable to common genetic variants.Peer reviewe

The Pentose Phosphate Pathway Regulates the Circadian Clock

The circadian clock is a ubiquitous timekeeping system that organizes the behavior and physiology of organisms over the day and night. Current models rely on transcriptional networks that coordinate circadian gene expression of thousands of transcripts. However, recent studies have uncovered phylogenetically conserved redox rhythms that can occur independently of transcriptional cycles. Here we identify the pentose phosphate pathway (PPP), a critical source of the redox cofactor NADPH, as an important regulator of redox and transcriptional oscillations. Our results show that genetic and pharmacological inhibition of the PPP prolongs the period of circadian rhythms in human cells, mouse tissues, and fruit flies. These metabolic manipulations also cause a remodeling of circadian gene expression programs that involves the circadian transcription factors BMAL1 and CLOCK, and the redox-sensitive transcription factor NRF2. Thus, the PPP regulates circadian rhythms via NADPH metabolism, suggesting a pivotal role for NADPH availability in circadian timekeeping.Peer reviewe

Ca2+-activated K+ channels reduce network excitability, improving adaptability and energetics for transmitting and perceiving sensory information

Ca2+-activated K+ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modelling, we discover how visual information transfer in mutants lacking the BK channel (dSlo-), SK channel (dSK-) or both (dSK-;;dSlo-) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K+ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants’ distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca2+-activated K+ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information

The pore structure and gating mechanism of K2P channels

K2P potassium channels are important regulators of cellular excitability. This study reveals that in contrast to most other K+ channels the primary gating mechanism in the K2P channel TREK-1 does not involve opening and closure of the cytoplasmic bundle crossing, but takes place close to or within the selectivity filter