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Constrained by The Matrix: Will Chloride Ions Be Set Free?

Commentary It is now well established that GABA receptors can mediate either excitation or inhibition, depending on the GABA reversal potential (EGABA) of the neuron, which in turn is set by the transmembrane chloride (Cl) gradient. In mature neurons, the Cl concentration outside the cell (Clo) is higher than that inside the cell, so when GABA binds to its postsynaptic type-A receptor and opens chloride channels, Cl flows down its electrochemical gradient into the cell, leading to hyperpolarization. In neonatal neurons, however, the intracellular Cl concentration (Cli) is high enough such that GABA binding-induced chloride channel opening leads to Cl efflux from the neuron, leading to membrane depolarization. Depolarizing GABA action early in development is considered to be important for neuronal plasticity and circuit maturation (1) and has also been used to explain the enhanced susceptibility of the neonatal brain to seizures (2, 3). Phenobarbital (PHB), a GABA receptor agonist, has limited effectiveness as an anticonvulsant in neonatal seizures, an effect that has been attributed to this reversed Cl gradient. In accordance with this mechanism, PHB not only fails to stop neonatal seizures but can even exacerbate them. Beyond the neonatal period, GABA depolarizing actions may have wider ramifications in epilepsy. For example, GABA induces depolarization in amygdala neurons in temporal lobe epilepsy (4). Two chloride cation cotransporters (CCCs) act in concert to govern a neuron’s Cl concentration gradient. The sodiumpotassium-chloride cotransporter 1 (NKCC1), predominates in the neonatal period and imports Cl into neurons. Later, expression of the potassium-chloride cotransporter 2 (KCC2) increases, with Cl transported out of the neuron. Thus, over development, KCC2 expression increases and NKCC1 expression decreases, such that when KCC2 predominates, the extracellular concentration of Cl is higher than its intracellular concentration. Until now, the explanation for the reversed chloride gradient and GABA-mediated depolarization early in development has implicated a developmental switch in the predominance of CCCs (3). Is the action of the CCCs sufficient to explain the relative differences in intraand extracellular Cl concentrations? The present paper by Glykys et al. proposes that CCC actions, while conceptually attractive, fail to account fully for Cli and the observed GABA equilibrium potential, EGABA. Evidence is presented that Cli sets EGABA but that CCC expression does not fully explain Cli and EGABA. If not CCC, what else could determine Cl concentration and thus the polarity of GABA action? Glykys et al. hypothesize that impermeant anions on both sides of the neuronal membrane contribute to Cl homeostasis. Intracellularly, impermeant anions consist of nucleotides with negatively charged phosphate groups and proteins with negatively charged amino and carboxyl groups. In the extracellular matrix, negatively charged sulfates on proteoglycans comprise the bulk of impermeant anions. Glykys et al. base their experiments on the assumption that the sum of intracellular anions and Cl must equal the sum of extracellular anions and Cl (Ai + Cli = Ao + Clo). There is a reciprocal relationship between Ai and Cli so that their sum must be constant—any increase in Cli must be balanced by a decrease in Ai, such that Cli is “constrained” by Ai. Similarly, in the extracellular matrix, Clo is constrained by Ao. To maintain Donnan equilibrium, an increase in either anion species within the intracellular or extracellular domain must balance. This reLocal Impermeant Anions Establish the Neuronal Chloride Concentration.