Sprecher
Beschreibung
Cation chloride cotransporters (CCCs) are evolutionary conserved proteins mediating the electroneutral transport of Cl-, K+ and/or Na+. While KCCs export chloride out of the cell, the opposing N(K)CCs function as chloride importers. Their activity is reciprocally regulated by phosphorylation through chloride-sensitive Wnk and SPAK/OSR1 (D.m.: Fray) kinases, resulting in tight regulation of the intracellular chloride level [Cl-]i. Normally, [Cl-]i is low, whereby binding of GABA to the ionotropic GABA-A receptor results in neuronal inhibition through chloride influx. However, when [Cl-]i is high, GABA can also exert an excitatory response. In the mammalian SCN expression and activity of NKCC and KCC underlie daily and regional variations. Resulting differences in GABA polarity have been shown to contribute to light-dependent synchronization of the circadian clock, e.g. phase delaying in response to a light pulse and adaption to seasonal changes in day length. Previous studies indicate similar mechanisms may operate in the fly clock system, where NKCC is known to influence light-input and GABA responses in clock neurons (Buhl et al. 2016 PNAS). Here we provide further evidence for the role of KCC and Wnk/Fray in regulating GABA responses and behavioral light responses (Eick et al. 2022 Current Biology). While wild type flies become arrhythmic under constant light (LL), up- or downregulation of KCC, Wnk and Fray led to robust locomotor rhythms in LL, indicating their requirement for normal light-input to the clock. Next, we measured the GABA-induced currents in l-LNv clock neurons to calculate the GABA reversal potential EGABA. As expected, KCC overexpression lowered EGABA, while kcc knockout resulted in a more positive EGABA, changing GABA into an excitatory neurotransmitter. Finally, we recorded the locomotor activity under long days. All flies were able to adapt to a longer photoperiod, but while NKCC knockout flies exhibited an abnormal increase in activity during the early morning, activity was notably reduced in kcc knockout flies at that time. All in all, our results suggest that regulation of [Cl-]i in clock neurons via CCCs is a conserved mechanism that is crucial for normal clock function.