Abstract
Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies.1,2 Circadian variation in the intracellular chloride concentration of mammalian pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels.3 However, results have been contradictory,4–9 and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response.10 Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLNv) pacemaker neurons. In control flies, intracellular chloride increases in sLNvs over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations.11 Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLNv pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1429-1438.e6 |
| Journal | Current Biology |
| Volume | 32 |
| Issue number | 6 |
| DOIs | |
| State | Published - Mar 28 2022 |
Funding
The authors would like to thank Billy Leiserson, Michael Rosbash, and Daria Hekmat-Scafe for fly lines; Johannes Bischof and Konrad Basler for plasmids; Helmut Krämer for S2-R+ cells; and Diana Lim for assistance with figures. Stocks obtained from the Bloomington Drosophila Stock Center ( NIH P40OD018537 ) and the Vienna Drosophila Resource Center were used in this study. Plasmids and S2-R+ cells were obtained from the Drosophila Genomics Research Center (Indiana University, Bloomington, IN, supported by NIH grant 2P40OD010949 ). The PDF C7 antibody, developed by Justin Blau (New York University), was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported by the National Institutes of Health : DK091316 , DK106350 , and DK110358 to A.R.R.; DK111542 to C.-L.H.; and AA019526 , AA026818 , and DA049635 to A.R. The authors would like to thank Billy Leiserson, Michael Rosbash, and Daria Hekmat-Scafe for fly lines; Johannes Bischof and Konrad Basler for plasmids; Helmut Krämer for S2-R+ cells; and Diana Lim for assistance with figures. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and the Vienna Drosophila Resource Center were used in this study. Plasmids and S2-R+ cells were obtained from the Drosophila Genomics Research Center (Indiana University, Bloomington, IN, supported by NIH grant 2P40OD010949). The PDF C7 antibody, developed by Justin Blau (New York University), was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported by the National Institutes of Health: DK091316, DK106350, and DK110358 to A.R.R.; DK111542 to C.-L.H.; and AA019526, AA026818, and DA049635 to A.R. Conceptualization, A.R.R.; investigation, J.N.S. Q.S. J.M.P. and S.-W.A.; formal analysis, A.R.R.; resources, J.H. and G.M.; writing – original draft, A.R.R.; writing – review & editing, A.R.R. I.T. and A.R.; visualization, A.R.R. and I.T.; supervision, A.R.R. and C.-L.H.; project administration, A.R.R.; funding acquisition, A.R.R. A.R. and C.-L.H. The authors declare no competing interests. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more of the authors of this paper self-identifies as living with a disability.
Keywords
- Drosophila
- Fray
- KCC
- NKCC
- SLC12
- SPAK
- WNK
- chloride signaling
- circadian rhythm
- potassium channel
ASJC Scopus subject areas
- General Biochemistry, Genetics and Molecular Biology
- General Agricultural and Biological Sciences