Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells

Marvin R Diaz,Benjamin A Hughes,C Fernando Valenzuela,Aya Wadleigh,John J Woodward

doi:10.3389/fnins.2011.00148

Marvin R Diaz, Benjamin A Hughes + Show 3 more

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https://doi.org/10.3389/fnins.2011.00148

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Abstract

The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca2+-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50–75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABAA-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl−-channel blockers were also tested: 4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs.

Highlights

The cerebellum controls motor coordination, cognitive function, and emotion
They are mediated in part by action potential-dependent mechanisms, as tetrodotoxin (TTX) can significantly reduce both synaptic GABA events driven by spontaneous firing of Golgi cells and the tonic current mediated by extrasynaptic GABAA receptors (GABAARs) (Kaneda et al, 1995; Brickley et al, 1996; Wall and Usowicz, 1997; Carta et al, 2004)
EFFECT OF ETHANOL ON RECOMBINANT BEST1 CHANNELS We initially investigated whether recombinant Best1 channels were sensitive to acute ethanol exposure

Summary

Introduction

The cerebellum controls motor coordination, cognitive function, and emotion. Its unique and well organized neuro-architecture is comprised of numerous layers each containing specific neurontypes that process incoming excitatory information from the brain stem and spinal cord. Numerous studies have characterized tonic currents in the cerebellum and have shown that these are sensitive to GABAA antagonists (Brickley et al, 1996; Carta et al, 2004; Bright et al, 2011) They are mediated in part by action potential-dependent mechanisms, as tetrodotoxin (TTX) can significantly reduce both synaptic GABA events driven by spontaneous firing of Golgi cells and the tonic current mediated by extrasynaptic GABAARs (Kaneda et al, 1995; Brickley et al, 1996; Wall and Usowicz, 1997; Carta et al, 2004)

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