NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

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, gamma-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 -30 mV. Because of the low HCO3- 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+-K+-2Cl (-) cotransporter, NKCC1, 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 HCO3- 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.