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

Shiu, F. H.; Dzhala, V. I.; Lillis, K.; Staley, K. J.

doi:10.64898/2026.09.24.753897

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bioRxiv · 10.64898/2026.09.24.753897

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

Shiu, F. H.·Dzhala, V. I.·Lillis, K.·Staley, K. J.

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Abstract

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.

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Shiu, F. H., Dzhala, V. I., Lillis, K., Staley, K. J.. 2026-09-30. Acute ClC-2 blockade increases neuronal intracellular chloride and depolarizes EGABA. https://doi.org/10.64898/2026.09.24.753897

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