Chloride fluxes and GABA release sustain inhibition in the CNS: The role for Bestrophin 1 anion channels

In the current issue of Acta Physiologica, Di Papma et al.¹ revealed a widespread brain expression of Ca²⁺-dependent anion (chloride) channel Bestrophin 1 (Best1) in both neurones and neuroglia. Chloride ions (Cl⁻) are indispensable for ionotropic inhibition of neurons in the central nervous system (CNS). This inhibition is mainly mediated by GABA_A and glycine pentameric receptors, the ligand-gated anion channels. Thus, controlling Cl⁻ homeostasis is paramount for balancing inhibition and excitation in the nervous circuits, which is critical for CNS function. An aberrant inhibition in the nervous circuits leads to many neurological and neuropsychiatric diseases, including epilepsy and mood disorders.^{2, 3}

Homeostasis of Cl⁻ in the CNS is functionally segregated between neurones and astrocytes. In the mature brain, neurones keep cytoplasmic Cl⁻ concentration ([Cl⁻]_i) low at around ~5–10 mM, while astrocytes maintain high [Cl⁻]_i in the range of 30–60 mM.⁴ This disparity defines the functional outcome of the opening of anion channels: in neurones an opening of anion channels mediates Cl⁻ influx (which results in hyperpolarization which inhibits neuronal activity), whereas in astrocytes these channels mediate depolarising Cl⁻ efflux. Such an opposite arrangement of the [Cl⁻]_i homeostasis is critical for maintaining synaptic and extrasynaptic neuronal inhibition. That is, Cl⁻ influx into neurones may deplete Cl⁻ from the extracellular space but Cl^- is replenished by a continuous supply of Cl⁻ ions from astrocytes.⁵ This coordinated Cl⁻ movement between cells and extracellular space is greatly facilitated by a close synaptic association of neuronal and astrocytic compartments, which form a multipartite synapse and a synaptic cradle.⁶ At the inhibitory synapses, the postsynaptic neuronal specialization, as well as astrocytic perisynaptic leaflets, possess GABA_A receptors.⁵ Hence, presynaptic GABA release opens anion channels in both neuronal and astrocytic membranes. Considering that extracellular Cl⁻ concentration can be less than the presumed 120 mM,⁷ astrocytic Cl⁻ supply is critical for sustaining inhibitory synaptic transmission. Indeed, optogenetic manipulations with astrocytic [Cl⁻]_i substantially affect neuronal inhibition.⁴

Another key player in Cl⁻ homeostasis in the brain tissue is represented by Ca²⁺-activated Cl⁻ channels that link together cells excitation, expressed as an intracellular Ca²⁺ raise, and transmembrane Cl⁻ flux that depolarises the membrane in astrocytes, and hyperpolarises neurones suppressing Ca²⁺ influx. Thus, Ca²⁺-activated Cl⁻ channels contribute to adjusting Cl⁻ flux, and hence coordinate synaptic Cl⁻ homeostasis with cellular activation state. The molecular origin of this Ca²⁺-activated Cl⁻ channel is debated, but Best1 protein, expressed in the CNS, is proposed to be a molecular substrate for Ca²⁺-dependent Cl⁻ flux in the brain.⁸ Bestrophin's ion-conducting properties were characterized in details electrophysiologically and pharmacologically,⁹ while crystallography revealed the conservative pore-forming structure of Best1.¹⁰ Of note, the ion conductance of Best1 is not strictly selective for Cl⁻ and it is permeable to a broad range of anions. Therefore, Best1 should rather be considered as the Ca²⁺-activated anion channels.

Bestrophins are not the only known Ca²⁺-activated Cl⁻ channel proteins. Several members of the TMEM16 protein family, TMEM16A and TMEM16B, also form Ca²⁺-activated Cl⁻ channel.¹¹ The reason for having two distinct Ca²⁺-activated Cl⁻ channel protein families is unclear, especially because these proteins are often shown to co-express in the same cells.¹² Arguably, difference in permeability for various anions may ascribe different cell functions to these channels. Their specific functions remain to be elucidated, but it has to be noted that the expression of TMEM16A and bestrophins is linked together providing another layer for interaction between these protein families.^{12, 13} It is, therefore, surprising that although Best1 expression and function were characterized in detail in astrocytes, the astrocytic TMEM16 did not, as yet, come under the spotlight. Furthermore, although Best1 has been implicated in the modulation of neuronal circuits, the absence of CNS phenotypes in retinal diseases linked to Best1 mutations remains puzzling. Further studies are required to determine if, and how, these mutations might influence circuit dynamics and brain function. The argument that astrocytes may compensate for the lack of Best1 function highlights the need to focus more closely on other Ca²⁺-activated anion/chloride channels, which may play a complementary or compensatory role in maintaining ion homeostasis in the CNS.

In recent years, Best1 channels received special attention in the studies of astrocytes, due to their ability to conduct not only Cl⁻ ions but also act as a conduit for two major neurotransmitters, glutamate, and GABA.¹⁴ Thus, Best1 channels are implicated in the tonic release of both neurotransmitters. In particular, astrocytic Best1 channels mediate tonic GABA inhibition in several brain regions, including the cerebellum and thalamus. The astrocytic tonic GABA inhibition in the thalamus is instrumental for the regulation of sensory acuity.¹⁵ Tonic GABA inhibition was also implicated in the pathophysiology of neurodegeneration, and Alzheimer's disease (AD) in particular. Both normal aging and AD-like pathology are associated with an increase in astrocytic GABA synthesis from putrescin through monoaminoxidase-B (MAO-B) catalyzed pathway, or through the urea cycle involved in the degradation of β-amyloid.¹⁶ The increased GABA production by astrocytes and consequent augmentation of tonic inhibition might, arguably, be a part of the defense response aimed to reduce neuronal hyperexcitability, a prominent feature of AD neuropathology.¹⁷ It is probably not a coincidence that a diffusional release of GABA through Best1 associates with Cl⁻ efflux from astrocytes to strengthen and sustain the tonic inhibition involving with continuous drainage of extracellular Cl⁻ ions to neurons. Astrocytic Best1 provides both the neurotransmitter and an inhibitory ion supplement, thus, reinforcing an effective and long-lasting inhibition of neurones. While accumulating evidence suggests astrocytes can release GABA, direct in vivo characterization is still lacking due to methodological challenges in achieving both specificity and sensitivity; advancing this line of research is essential to reveal how astrocytic GABA dynamics shape neuronal circuitry in the intact brain.

Astrocytes are not, however, the only possessors of Best1. The paper by Fiorenzo Conti, Justin Lee, and their colleagues published in Acta Physiologica¹ showed that neuronal expression of Best1 is comparable with that in astrocytes. Best1 was also shown to express in oligodendroglia and microglia, but at substantially smaller quantities. Moreover, neuronal Best1 channels demonstrated a peculiar distribution being concentrated in GABAergic presynaptic terminals and, to a lesser extent, in glutamatergic presynaptic compartments. This finding further widens and diversifies the possible contribution of Best1 to inhibitory and excitatory neurotransmission, both phasic and tonic. It seems that both glutamate and GABA can be released from presynaptic terminals in a non-vesicular manner in response to presynaptic Ca²⁺ signals opening the Best1 channels. The magnitude of a channel-mediated, diffusion-driven release of neurotransmitters is tightly controlled by their cytosolic concentration, which in neurones is quite high (mM range), arguably much higher than in astrocytes, where glutamate is limited by glutamine synthetase, whereas GABA concentration is relatively low because of less effective synthesis and utilization in the Krebs cycle.¹⁸ At the same time, however, neuronal Best1 channels mediate Cl⁻ influx, hyperpolarising the presynaptic terminal and reducing neurotransmitter exocytosis. Although cell type-specific manipulations have largely defined the role of Best1 in astrocytes and brain function, findings from studies employing generalized approaches warrant careful re-evaluation and interpretation. Thus, complex Best1-mediated fluxes of neurotransmitters and Cl⁻, which involve both astrocytes and neurones (Figure 1) should be considered when analyzing inhibitory and excitatory neurotransmission in the CNS.

The authors contributed equally to this editorial.