Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade

Jeffrey N. Schellinger, Qifei Sun, John M. Pleinis, Sung Wan An, Jianrui Hu, Gaëlle Mercenne, Iris Titos, Chou Long Huang, Adrian Rothenfluh, Aylin R. Rodan

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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 languageEnglish (US)
Pages (from-to)1429-1438.e6
JournalCurrent Biology
Issue number6
StatePublished - Mar 28 2022
Externally publishedYes


  • 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


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