Circadian Rhythm Regulation by Pacemaker Neuron Chloride Oscillation in Flies

Abstract

Circadian rhythms in physiology and behavior sync organisms to external environmental cycles. Here, circadian oscillation in intracellular chloride in central pacemaker neurons of the fly, Drosophila melanogaster, is reviewed. Intracellular chloride links SLC12 cation-coupled chloride transporter function with kinase signaling and the regulation of inwardly rectifying potassium channels.

Keywords: SLC12; SPAK; WNK; circadian; potassium channel.

FIGURE 1.

Initial model explaining the role of intracellular chloride in mammalian suprachiasmatic nucleus (SCN) pacemaker neurons Most cells have the sodium-potassium ATPase (Na⁺/K⁺-ATPase), which uses energy from ATP to pump 3 sodium (Na⁺) out of cells and 2 potassium (K⁺) in. As a result, intracellular sodium and extracellular potassium concentrations are low, creating the chemical gradients for chloride entry via cotransport through sodium-potassium-2-chloride cotransporters (NKCC) and for chloride exit via cotransport through potassium-chloride cotransporters (KCC). In conditions of high NKCC activity and/or low KCC activity, intracellular chloride will accumulate in cells, whereas in conditions of low NKCC activity and/or high KCC activity, intracellular chloride concentrations will be low. In 1997, Wagner et al. (7) proposed that high intracellular chloride during the day would result in excitatory effects of GABAergic signaling onto SCN pacemaker neurons, since chloride efflux through the GABA_A receptor ligand-gated chloride channel would result in membrane depolarization. In contrast, low nighttime intracellular chloride concentrations will result in chloride influx through the GABA_A receptor chloride channel, membrane hyperpolarization, and decreased SCN neuron activity.

FIGURE 2.

Increased intracellular chloride inhibits WNK-Fray activation of the Irk1 potassium channel to regulate circadian period A: at zeitgeber time (ZT)2, intracellular chloride in the Drosophila sLN_v (small ventrolateral neurons) central pacemaker neurons is low due to low activity of the NKCC encoded by Ncc69. Chloride inhibits with no lysine (K) (WNK) kinases. In low chloride conditions, WNK will be active and phosphorylate and activate the downstream kinase, Fray. Fray in turn activates the inwardly rectifying potassium channel Irk1. B: at ZT6, chloride entry via the NKCC encoded by Ncc69 over the course of the morning restrains WNK-Fray signaling and Irk1 activity. Ncc69 mutants remain “stuck” with low intracellular chloride, as seen normally at ZT2. C and D: disruption of this pathway results in disruptions of circadian locomotor rhythms in both light/dark conditions (C) and in constant darkness (D). C: locomotor activity of controls vs. Ncc69^r2 mutants, showing loss of morning anticipation (the anticipatory increase in locomotor activity before lights on, arrow). D: representative actograms showing average activity across 7 days of subjective day (gray) and night (dark) in constant darkness, in which the free-running clock is operative. Ncc69^r2 mutants have a prolonged circadian period. E: resting membrane potential was measured using whole cell recordings of the sLN_v pacemaker neurons and analyzed using linear regression analysis. Resting membrane potential becomes more hyperpolarized from ZT0 (lights-on) to ZT6, is variable between ZT6 and ZT18, and becomes depolarized between ZT18 and ZT24/ZT0. Changes in Irk1 activity due to changes in WNK-Fray signaling could affect resting membrane potential and neuronal excitability. For example, in the presence of unrestrained WNK-Fray signaling, as would occur with loss of the Ncc69-encoded NKCC, Irk1 would remain activated at ZT6, which could keep the resting membrane potential in a hyperpolarized state and prevent subsequent depolarization before morning. A–D are adapted or reproduced from Ref. , with permission from Elsevier. E is reproduced from Ref. with permission from Journal of Neuroscience.

FIGURE 3.

How do circadian oscillations in intracellular chloride in Drosophila and mammalian central pacemaker neurons interface with the molecular clock? A: simplified schematics of Drosophila and mammalian transcriptional-translational feedback loops are shown. In Drosophila, heterodimeric CLOCK (CLK)-CYCLE (CYC) activates transcription of period (per) and timeless (tim). The protein products PER and TIM then feedback repress CLK-CYC. A similar loop is operative in mammals. BMAL1 is the ortholog of CYC and CRY (CRYPTOCHROME) replaces TIM. Not shown are the multiple paralogs of mammalian PER and CRY, as well as additional transcriptional-translational feedback loops and other regulatory mechanisms that are operative in both systems. The connections between the core clock and intracellular chloride oscillations are currently unknown. B and C: long-term imaging of chloride every 5 or 10 minutes over 4 days was performed in cultured explants of the suprachiasmatic nucleus in arginine vasopressin-expressing (B) and vasoactive intestinal peptide-expressing neurons (C), using the ratiometric chloride sensor Cl-Sensor. Data were detrended in the Lumicycle Analysis Program (Actimetrics) using the 24 hour rolling average baseline subtraction. Lights on is at zeitgeber time (ZT)24, ZT48, ZT72, and ZT96, with a higher ratio indicating higher intracellular chloride concentrations. Thus intracellular chloride rises during the day and falls at night. B and C are reproduced from Ref. , with permission from Nathan J. Klett, Olga Cravetchi, and Charles N. Allen.