WNK3 and WNK4 Exhibit Opposite Sensibility for Cell Volume and Intracellular Chloride Concentration

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl−]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl−]i, respectively. Cell shrinkage and a decrease in [Cl−]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl−]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl−]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl−]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl−]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK’s carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.

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Long-Term Imaging Reveals a Circadian Rhythm of Intracellular Chloride in Neurons of the Suprachiasmatic Nucleus

Journal of Biological Rhythms ◽

10.1177/07487304211059770 ◽

2022 ◽

pp. 074873042110597

Author(s):

Nathan J. Klett ◽

Olga Cravetchi ◽

Charles N. Allen

Keyword(s):

Suprachiasmatic Nucleus ◽

Chloride Concentration ◽

Intracellular Chloride ◽

Voltage Gated Calcium Channels ◽

Excitatory Gaba ◽

Ratiometric Probe ◽

Gaba Transmission ◽

Circadian Physiology ◽

Intracellular Chloride Concentration

Both inhibitory and excitatory GABA transmission exist in the mature suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. Whether GABA is inhibitory or excitatory depends on the intracellular chloride concentration ([Cl−]i). Here, using the genetically encoded ratiometric probe Cl-Sensor, we investigated [Cl−]i in AVP and VIP-expressing SCN neurons for several days in culture. The chloride ratio (RCl) demonstrated circadian rhythmicity in AVP + neurons and VIP + neurons, but was not detected in GFAP + astrocytes. RCl peaked between ZT 7 and ZT 8 in both AVP + and VIP + neurons. RCl rhythmicity was not dependent on the activity of several transmembrane chloride carriers, action potential generation, or the L-type voltage-gated calcium channels, but was sensitive to GABA antagonists. We conclude that [Cl−]i is under circadian regulation in both AVP + and VIP + neurons.

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GABA-mediated repulsive coupling between circadian clock neurons in the SCN encodes seasonal time

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421200112 ◽

2015 ◽

Vol 112 (29) ◽

pp. E3920-E3929 ◽

Cited By ~ 77

Author(s):

Jihwan Myung ◽

Sungho Hong ◽

Daniel DeWoskin ◽

Erik De Schutter ◽

Daniel B. Forger ◽

...

Keyword(s):

Circadian Clock ◽

Synaptic Input ◽

Chloride Concentration ◽

Phase Relationship ◽

Day Length ◽

Intracellular Chloride ◽

Circadian Oscillators ◽

Length Changes ◽

Excitatory Gaba ◽

Intracellular Chloride Concentration

The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABAA-mediated synaptic input. We show that increasing day-length changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABAA signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation–inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.

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Influence of WNK3 on intracellular chloride concentration and volume regulation in HEK293 cells

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-012-1137-4 ◽

2012 ◽

Vol 464 (3) ◽

pp. 317-330 ◽

Cited By ~ 15

Author(s):

Silvia Cruz-Rangel ◽

Gerardo Gamba ◽

Gerardo Ramos-Mandujano ◽

Herminia Pasantes-Morales

Keyword(s):

Volume Regulation ◽

Chloride Concentration ◽

Hek293 Cells ◽

Intracellular Chloride ◽

Intracellular Chloride Concentration

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Intracellular Chloride Concentration and Evidence for the Existence of a Chloride Pump in Frog Skeletal muscle

The Japanese Journal of Physiology ◽

10.2170/jjphysiol.30.357 ◽

1980 ◽

Vol 30 (3) ◽

pp. 357-363 ◽

Cited By ~ 6

Author(s):

Tetsuji HIRONAKA ◽

Shoji MORIMOTO

Keyword(s):

Skeletal Muscle ◽

Chloride Concentration ◽

Frog Skeletal Muscle ◽

Intracellular Chloride ◽

Chloride Pump ◽

Intracellular Chloride Concentration

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GABAA-Dependent Chloride Influx Modulates Reversal Potential of GABAB-Mediated IPSPs in Hippocampal Pyramidal Cells

Journal of Neurophysiology ◽

10.1152/jn.2001.85.6.2381 ◽

2001 ◽

Vol 85 (6) ◽

pp. 2381-2387

Author(s):

Valeri Lopantsev ◽

Philip A. Schwartzkroin

Keyword(s):

Membrane Potential ◽

Mossy Fiber ◽

Chloride Concentration ◽

Reversal Potential ◽

Pyramidal Cells ◽

Intracellular Chloride ◽

The Impact ◽

Intracellular Chloride Concentration ◽

Chloride Influx ◽

Hippocampal Pyramidal Cells

Changes in intracellular chloride concentration, mediated by chloride influx through GABAA receptor–gated channels, may modulate GABAB receptor–mediated inhibitory postsynaptic potentials (GABAB IPSPs) via unknown mechanisms. Recording from CA3 pyramidal cells in hippocampal slices, we investigated the impact of chloride influx during GABAA receptor–mediated IPSPs (GABAA IPSPs) on the properties of GABAB IPSPs. At relatively positive membrane potentials (near −55 mV), mossy fiber–evoked GABAB IPSPs were reduced (compared with their magnitude at −60 mV) when preceded by GABAAreceptor–mediated chloride influx. This effect was not associated with a correlated reduction in membrane permeability during the GABAB IPSP. The mossy fiber–evoked GABAB IPSP showed a positive shift in reversal potential (from −99 to −93 mV) when it was preceded by a GABAA IPSP evoked at cell membrane potential of −55 mV as compared with −60 mV. Similarly, when intracellular chloride concentration was raised via chloride diffusion from an intracellular microelectrode, there was a reduction of the pharmacologically isolated monosynaptic GABABIPSP and a concurrent shift of GABAB IPSP reversal potential from −98 to −90 mV. We conclude that in hippocampal pyramidal cells, in which “resting” membrane potential is near action potential threshold, chloride influx via GABAA IPSPs shifts the reversal potential of subsequent GABAB receptor–mediated postsynaptic responses in a positive direction and reduces their magnitude.

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Shift of Intracellular Chloride Concentration in Ganglion and Amacrine Cells of Developing Mouse Retina

Journal of Neurophysiology ◽

10.1152/jn.00578.2005 ◽

2006 ◽

Vol 95 (4) ◽

pp. 2404-2416 ◽

Cited By ~ 47

Author(s):

Ling-Li Zhang ◽

Hemal R. Pathak ◽

Douglas A. Coulter ◽

Michael A. Freed ◽

Noga Vardi

Keyword(s):

Gaba Receptors ◽

Chloride Concentration ◽

Reversal Potential ◽

Amacrine Cells ◽

Resting Potential ◽

Equilibrium Potential ◽

Mouse Retina ◽

Intracellular Chloride ◽

Excitatory Action ◽

Intracellular Chloride Concentration

GABA and glycine provide excitatory action during early development: they depolarize neurons and increase intracellular calcium concentration. As neurons mature, GABA and glycine become inhibitory. This switch from excitation to inhibition is thought to result from a shift of intracellular chloride concentration ([Cl−]i) from high to low, but in retina, measurements of [Cl−]i or chloride equilibrium potential ( ECl) during development have not been made. Using the developing mouse retina, we systematically measured [Cl−]i in parallel with GABA's actions on calcium and chloride. In ganglion and amacrine cells, fura-2 imaging showed that before postnatal day (P) 6, exogenous GABA, acting via ionotropic GABA receptors, evoked calcium rise, which persisted in HCO3−- free buffer but was blocked with 0 extracellular calcium. After P6, GABA switched to inhibiting spontaneous calcium transients. Concomitant with this switch we observed the following: 6-methoxy- N-ethylquinolinium iodide (MEQ) chloride imaging showed that GABA caused an efflux of chloride before P6 and an influx afterward; gramicidin-perforated-patch recordings showed that the reversal potential for GABA decreased from −45 mV, near threshold for voltage-activated calcium channel, to −60 mV, near resting potential; MEQ imaging showed that [Cl−]i shifted steeply around P6 from 29 to 14 mM, corresponding to a decline of ECl from −39 to −58 mV. We also show that GABAergic amacrine cells became stratified by P4, potentially allowing GABA's excitatory action to shape circuit connectivity. Our results support the hypothesis that a shift from high [Cl−]i to low causes GABA to switch from excitatory to inhibitory.

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