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NKCC Regulates Circadian Rhythm Via the Chloride‐Sensing WNK Kinase
Author(s) -
Rodan Aylin,
Schellinger Jeffrey,
Sun Qifei,
Rothenfluh Adrian
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1007.12
Subject(s) - circadian rhythm , medicine , endocrinology , period (music) , drosophila melanogaster , mutant , cotransporter , wild type , intracellular , biology , protein kinase a , chemistry , sodium , kinase , microbiology and biotechnology , biochemistry , gene , physics , organic chemistry , acoustics
Objective Oscillation of intracellular Cl − has been proposed to play a role in circadian rhythms. We examined the role of Cl − transporters and the Cl − ‐sensitive WNK (with‐no‐lysine) kinase in the regulation of circadian rhythm in Drosophila melanogaster.Methods We measured the circadian period of wild‐type and mutant flies using 24‐hour activity monitoring. We measured chloride concentration in the Drosophila brain pacemaker neurons using the transgenic sensor, ChlopHensor. Results were analyzed in GraphPad Prism using t‐test or one‐way ANOVA with Bonferroni correction for multiple comparisons. Results Male flies with null mutations in the Cl − influxing sodium‐potassium‐2‐chloride cotransporter, NKCC (encoded by the gene Ncc69 in flies), have an increased period length compared to controls (mean±SEM: 25.18±0.28 vs. 23.98±0.05 hours, p<0.0001) which is rescued by expression of wild‐type NKCC in the pacemaker neurons (23.78±0.08, p<0.0001 vs NKCC mutant). In NKCC mutant flies, knockdown of the Cl − effluxing potassium‐chloride cotransporter, KCC , in the pacemaker neurons also normalizes period length (23.94±0.27 vs 26.38±0.23 hours, p<0.0001). In control flies, Cl − concentration increased in pacemaker neurons between two hours after lights on (ZT2) and ZT6, from 44±7 to 81±8 mM, whereas in NKCC mutant flies, Cl − did not increase (35±6 to 44±8 mM), resulting in abnormally low intracellular Cl − concentration at ZT6 in the NKCC mutant flies compared to controls (p=0.0027). Cl − inhibits WNK. The NKCC mutant period length phenotype (26.67±0.13 h) was suppressed by knockdown of either wnk (24.70±0.29, p<0.0001) or its downstream kinase fray (homolog of mammalian SPAK/OSR1) (24.34±0.15, p<0.0001) in the pacemaker cells. Conversely, overexpression of a Cl − ‐insensitive (activated) wnk (25.34±0.10 h, p=0.0002 vs control) or constitutively active fray (25.56±0.26, p<0.0001 vs control), resulted in increased period length. Conclusion Normal activity of the fly NKCC is required to allow an increase in intracellular Cl − in the fly pacemaker neurons between two hours after lights on and six hours. The failure of intracellular Cl − to increase in NKCC mutant flies results in aberrant activation of WNK‐SPAK/OSR1 signaling and prolonged circadian period. Support or Funding Information National Institutes of Health