Biology subjects

Dzhala, V. I.

Publications and source records attributed to Dzhala, V. I..

3 recordsLinked to original sources

bioRxiv2026

Acute ClC-2 blockade increases neuronal intracellular chloride and depolarizes EGABA

Neuronal intracellular chloride concentration shapes the polarity and strength of GABAA receptor-mediated inhibition. ClC-2 is a hyperpolarization-activated chloride channel proposed to support chloride extrusion, but its acute contribution to EGABA and neuronal chloride homeostasis remains incompletely understood. Here, we used the selective ClC-2 inhibitor AK-42 to test how acute ClC-2 inhibition affects GABAergic chloride signaling in organotypic hippocampal slice cultures. We combined targeted single-interneuron optogenetic activation of stCoChR-expressing GABAergic interneurons with gramicidin-perforated patch recordings to measure optogenetically evoked inhibitory postsynaptic currents and calculate EGABA and we used SuperClomeleon imaging to measure neuronal intracellular chloride. AK-42 depolarized EGABA, with a larger effect when stimulation was delivered 2 s rather than 100 ms after voltage-step onset. This delay-dependent effect is consistent with greater engagement of voltage- and time-dependent ClC-2 activity during prolonged voltage steps. AK-42 also abolished the more hyperpolarized EGABA normally measured at the 2-s stimulation delay and shifted baseline holding currents at hyperpolarized command potentials without measurably changing estimated resting membrane potential. SuperClomeleon imaging showed that AK-42 increased neuronal intracellular chloride in the presence of TTX, supporting a role for ClC-2 in basal chloride regulation. Together, these findings show that acute ClC-2 inhibition depolarizes EGABA, increases neuronal intracellular chloride, and alters the temporal dynamics of GABAergic chloride signaling, identifying ClC-2 as an acute regulator of synaptic GABAergic chloride signaling.

neuroscience↗

bioRxiv2026

WNK-SPAK/OSR1 signaling pathway facilitates ictal activity via reduced neuronal chloride extrusion rate

Seizures upregulate Na+-K+-2Cl- (NKCC1)-mediated Cl- influx and downregulate K+-Cl- (KCC2)-mediated Cl- efflux via the WNK-SPAK/OSR1 kinases, leading to cytoplasmic chloride ([Cl-]i) accumulation, reduced GABAergic inhibition and anticonvulsant failure. Early studies found that inhibiting WNK-kinase reduced baseline [Cl-]i (ECl) and seizures via increased KCC2 activity. However, increased KCC2 activity alone should not affect ECl whose determinants are more complex. We determined the net effects of WNK-SPAK/OSR1 pathway inhibitor WNK463 on ECl and [Cl-]i transients during spontaneous ictal-like discharges (ILDs). We found that WNK463 reduced interictal [Cl-]i but did not change baseline [Cl-]i measured in the presence of TTX. WNK463 enhanced neuronal Cl- extrusion during and after ILDs, before abolishing ILDs. Pharmacological inhibition and targeted siRNA silencing demonstrated that the anti-ictal effects of WNK463 involved both NKCC1 and KCC2. Our data support a mechanism whereby mutual NKCC1 inhibition and KCC2 activation via the WNK-SPAK/OSR1 pathway exert powerful anti-ictal effects by facilitating [Cl-]i extrusion during ILDs.

neuroscience↗

bioRxiv2020

Unique actions of GABA arising from cytoplasmic chloride microdomains

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported, and we found a corresponding variance in the reversal potential (EGABA) for individual interneurons synapsing on a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl- ([Cl-i]). We determined [Cl-]i by: 1) two-photon imaging of the Cl- sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl- sensitive fluorophore MEQ; and 3) electrophysiological measurements of EGABA. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vivo. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs) but steadily decreased after inhibiting the polymerization of the anionic macromolecule actin. These studies highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs.

neuroscience↗