NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

Abstract

Gonzalez-Islas C, Chub N, Wenner P. NKCC1 and AE3 appear to accumulate chloride in embryonic motoneurons. J Neurophysiol 101: 507-518, 2009. First published November 26, 2008 doi:10.1152/jn.90986.2008. During early development, ␥-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (E GABA ) for GABA A miniature postsynaptic currents. When whole cell patch solutions contained 17-52 mM chloride, we found that synaptic E GABA was around Ϫ30 mV. Because of the low HCO 3 Ϫ permeability of the GABA A receptor, this value of E GABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized E GABA and reduced nerve potentials evoked by local application of a GABA A agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO 3 Ϫ and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons. I N T R O D U C T I O NA great deal of interest has recently been focused on the observation that neurons are depolarized by ␥-aminobutyric acid (GABA) in early development in several different parts of the nervous system (Ben-Ari et al. 2007). In this developmental period, the reversal potential for GABA A receptor currents (referred to as E GABA ) is more depolarized than the resting membrane potential because chloride, the main carrier of the GABA A current, is accumulated in immature neurons. The depolarizing nature of GABA is important in driving the spontaneous network activity (SNA) that is observed in virtually all developing circuits The modulation of intracellular chloride appears to be critical for the normal expression of SNA (Chub and O'Donovan 1998, 2001; Chub et al. 2006; Fedirchuk et al. 1999; Marchetti et al. 2005). During an episode of SNA, intracellular chloride is depleted as it passes out of the cell through activated GABA A receptor channels, thus weakening the driving force for GABAergic synapses, leaving the cord relatively less excitable. Then in the quiescent...

Cystic fibrosis transmembrane conductance regulator modulates synaptic chloride homeostasis in motoneurons of the rat spinal cord during neonatal development

“…It is noteworthy that the E GABA/Gly was less negative at P1 (À73.5 6 0.8 mV) than at P8 (À79.6 6 1.1 mV), and that in either case such a value was more negative than the E Cl calculated with the Nernst equation. The discrepancy between calculated E Cl and measured E GABA/Gly in the spinal cord has already been reported by JeanXavier et al (2006) and Gonzalez-Islas et al (2009). One parsimonious interpretation is that the intracellular Cl À concentration at synaptic level was kept at a relatively low level by a continuous extrusion, probably due to the operation of KCC2 that was already expressed at birth as shown in the present study.…”

“…In the presence of kynurenic acid to suppress ionotropic glutamate receptor and nicotinic cholinergic events (Hilmas et al, 2001;Marchetti et al, 2002), we recorded E GABA/Gly of synaptic currents, a parameter that provides a reliable index of Cl À concentration at the synaptic level (Gonzalez-Islas et al, 2009). Of course, such an experimental approach cannot provide information on the Cl À homeostasis throughout different cell compartments.…”

“…By inhibiting CFTR, we might have perturbed this equilibrium mainly when NKCC1 is allegedly most active, namely at birth (Ben-Ari, 2002). Indeed, a low concentration of bumetanide, which is reported to be a selective blocker of NKCC1 (Gonzalez-Islas et al, 2009), could significantly shift E GABA/Gly only at P1. Previous investigations have indicated a close interrelationship between CFTR and NKCC1 because, in epithelial cell cultures, CFTR stimulates NKCC1 to increase intracellular Cl À (Adam et al, 2005), as well as to enhance the functional expression of this transporter (Shumaker and Soleimani, 1999).…”

Non–cell-autonomous factor induces the transition from excitatory to inhibitory GABA signaling in retina independent of activity

“…However, this study does not address the mechanism by which this factor regulates intracellular chloride concentration. Possible mechanisms include regulation of a large number of proteins, including cation-chloride cotransporters (12), namely the Na + -K + -2Cl − cotransporters that increase [Cl − ] i (10,(31)(32)(33)(34)(35) (42), and a variety of chloride channels (13). In contrast to the hippocampus, RGCs do not express Na + -K + -2Cl − cotransporter 1 (23) and they do not express Cl − /HCO 3 + exchangers until later in development (43).…”

“…The transition from excitatory to inhibitory action of GABA has been observed in a number of neural circuits and in many vertebrate species (5,6) and the primary mechanisms underlying the switch are fairly well understood (7)(8)(9)(10)(11); reviewed in refs. [12][13][14].…”