Neuronal chloride concentration [Cl−]i is an important determinant of GABAA receptor (GABAAR)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCC) move Cl− across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl−]i. Measurement of [Cl−]i in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A]i) and polyanionic extracellular matrix glycoproteins ([A]o) constrain the local [Cl−]. CCC inhibition had modest effects on [Cl−]i and neuronal volume, but substantial changes were produced by alterations of the balance between [A]i and [A]o. Therefore, CCC are important elements of Cl− homeostasis, but local impermeant anions determine the homeostatic set-point for [Cl−], and hence, neuronal volume and the polarity of local GABAAR signaling.

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) alpha-II spectrin could assemble heterodimers with beta-II spectrin, but alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II (mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of alpha/beta spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy.

Angelman syndrome is a neurodevelopmental disorder caused by loss of function of the UBE3A gene encoding a ubiquitin E3 ligase. Motor dysfunction is a characteristic feature of Angelman syndrome, but neither the mechanisms of action nor effective therapeutic strategies have yet been elucidated. We report that tonic inhibition is specifically decreased in cerebellar granule cells of Ube3a-deficient mice, a model of Angelman syndrome. As a mechanism underlying this decrease in tonic inhibition, we show that Ube3a controls degradation of γ-aminobutyric acid (GABA) transporter 1 (GAT1) and that deficiency of Ube3a induces a surplus of GAT1 that results in a decrease in GABA concentrations in the extrasynaptic space. Administering low doses of 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP), a selective extrasynaptic GABA(A) receptor agonist, improves the abnormal firing properties of a population of Purkinje cells in cerebellar brain slices and reduces cerebellar ataxia in Ube3a-deficient mice in vivo. These results suggest that pharmacologically increasing tonic inhibition may be a useful strategy for alleviating motor dysfunction in Angelman syndrome.

Pharmaco-resistant seizures and cytotoxic cerebral edema are serious complications of ischemic and traumatic brain injury. Intraneuronal Cl− concentration ([Cl−]i) regulation impacts both cell volume homeostasis and Cl− permeable GABAA receptor-dependent membrane excitability. Understanding the pleiotropic molecular determinants of neuronal [Cl−]i –cytoplasmic impermeant anions, polyanionic extracellular matrix (ECM) glycoproteins, and plasmalemmal Cl− transporters– could help identify novel anti-convulsive and neuroprotective targets. The cation-Cl− cotransporters and ECM metalloproteinases may be particularly druggable targets for intervention. Here, we establish a paradigm that accounts for recent data regarding the complex regulatory mechanisms of neuronal [Cl−]i and how these mechanisms impact neuronal volume and excitability. We propose approaches to modulate [Cl−]i that are relevant for two common clinical sequela of brain injury: edema and seizures.