A Furosemide-Sensitive K⁺–Cl⁻Cotransporter Counteracts Intracellular Cl⁻Accumulation and Depletion in Cultured Rat Midbrain Neurons

Abstract

Efficacy of postsynaptic inhibition through GABA A receptors in the mammalian brain depends on the maintenance of a Cl Ϫ gradient for hyperpolarizing Cl Ϫ currents. We have taken advantage of the reduced complexity under which Cl Ϫ regulation can be investigated in cultured neurons as opposed to neurons in other in vitro preparations of the mammalian brain. GABA is the main inhibitory transmitter in the mammalian brain. The dominant effect of GABA A receptor activation is a hyperpolarization caused by C l Ϫ flux into the cell (for review, see Sivilotti and Nistri, 1991;Kaila, 1994;Thompson, 1994). However, the direction of the C l Ϫ flux depends on the C l Ϫ gradient across the membrane. Indeed, GABA A receptor-mediated hyperpolarizing and /or depolarizing postsynaptic potentials have been observed (for review, see Kaila, 1994;Thompson, 1994). Some findings suggest variations in intracellular [C l Ϫ ] between different neurons and even a distinct C l Ϫ distribution in different compartments of a single neuron (Misgeld et al., 1986). Depolarizing GABA A responses, however, can be caused by bicarbonate efflux in combination with C l Ϫ influx or combined Cl Ϫ and HCO 3 Ϫ efflux (Grover et al., 1993;Kaila, 1994;Thompson, 1994;Staley et al., 1995;Perkins and Wong, 1996;Kaila et al., 1997). To be able to predict the direction of C l Ϫ currents flowing during GABA A receptor-mediated inhibition it is essential to understand the regulation of C l Ϫ homeostasis that provides the transmembrane gradient.A recently cloned K ϩ -Cl Ϫ cotransporter gene (KCC2) represents a perfect candidate for the regulation of neuronal Cl Ϫ homeostasis (Payne et al., 1996). In contrast to the ubiquitous presence of the K ϩ -Cl Ϫ cotransporter KCC1, expression of the K ϩ -Cl Ϫ cotransporter KCC2 is detected in C NS only and seems to be neuron specific. KCC2 is also distinct from KCC1 in that KCC2 is not involved in cell volume regulation and not activated by osmotic changes. Furthermore, KCC2 has a high affinity for extracellular K ϩ ions. The properties of KCC2 allow the regulation of [Cl Ϫ ] i to maintain Cl Ϫ gradients for hyperpolarizing GABAergic inhibition. Thermodynamic considerations predict that the electroneutral K ϩ -Cl Ϫ cotransporter KCC2 operates near equilibrium under physiological ionic conditions. Depending on [Cl Ϫ ] i and [K ϩ ] o (Payne, 1997), the transport will extrude or accumulate Cl Ϫ .The functional role of a particular Cl Ϫ transport system in neuronal Cl Ϫ regulation is difficult to establish in studies using integral preparations such as brain slices. One complicating factor is the presence of HCO 3 Ϫ anions. The HCO 3 Ϫ permeability of the GABA A channel (Bormann, 1988;Fatima-Shad and Barry, 1993) impedes conclusions toward actual [Cl Ϫ ] i if they are calculated from reversal potentials of GABA A receptor-mediated anion currents. Furthermore, a HCO 3 Ϫ /Cl Ϫ exchanger (RaleySusman et al., 1993) may well interfere (Chesler, 1990), and pH changes that result from manipulations of [HCO 3 Ϫ ] o strongly affect neur...

Theta-bursts induce a shift in reversal potentials for GABA-A receptor-mediated postsynaptic currents in rat hippocampal CA1 neurons

“…Furosemide, when tested on 3 control neurons (in which no theta-bursts were given) did not significantly changed the RP of the IPSC (before furosemide application: 63.17 ± 0.59 mV & after furosemide application: 61.92 ± 0.46 mV; P > 0.05). This observation is consistent with those of (Banks et al, 1998;Jarolimek et al, 1996;Jarolimek et al, 1999;Ouardouz and Sastry, 2005;Pearce, 1993).…”

“…Furosemide, when tested on 3 control neurons (in which no theta-bursts were given) did not significantly changed the RP of the IPSC (before furosemide application: 63.17 ± 0.59 mV & after furosemide application: 61.92 ± 0.46 mV; P > 0.05). This observation is consistent with those of (Banks et al, 1998;Jarolimek et al, 1996;Jarolimek et al, 1999;Ouardouz and Sastry, 2005;Pearce, 1993).Previous studies in our laboratory indicated that tetanic stimulations of inputs induce a shift in the RP for GABA-ergic PSCs of neonatal rat DCN neurons (Ouardouz and Sastry ., 2000;Ouardouz and Sastry., 2005) and neonatal rat hippocampal CA1 pyramidal neurons (Ouardouz et al, 2006). In DCN neurons, the tetanus-induced negative shift in the RP seems to be due to an increased expression and activation of KCC-2 through an activation of protein kinase A, protein synthesis and activation of protein phosphatases (Ouardouz and Sastry, 2005).…”

Cation–chloride cotransporters mediate neural computation in the retina

“…Elsewhere in the adult CNS, GABA A and GABA C receptor activation can evoke both depolarizing and hyperpolarizing postsynaptic potentials from the same neuron (31). Moreover, different compartments of a single neuron have been reported to contain different intracellular chloride concentrations (32,33) in a manner that is consistent with the differential distribution of the Na-K-Cl and K-Cl cotransporters in the membranes of the proximal and distal portions of the dendrites, respectively. In addition, SAC dendrites exhibit a differential distribution of synaptic proteins because synaptic specializations are present on the distal, and not proximal, portions of their dendrites (20,27,28).…”

“…Finally, the results suggest that subcellular specializations, such as the differential distribution of the Na-K-Cl and K-Cl cotransporters, can mediate the neural computation of complex information. As occurs in the retina, it is likely that the differential distribution of the Na-K-Cl and K-Cl cotransporters may also mediate neural computations elsewhere in the brain, such as in the midbrain and hippocampus, where the processes of individual neurons exhibit a chloride gradient (32,33). Indeed, the asymmetric release of GABA from interneurons in the brain at dendrodendritic synapses may be an important means by which the brain processes information.…”

Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

“…Indeed, control measurements of somatic E GABA yielded a value of Ϫ40.7 Ϯ 0.41 mV (n ϭ 26), demonstrating that the somatic [Cl Ϫ ] i of CA1 pyramidal neurons was clamped at the [Cl Ϫ ] of the pipette. Thus, under these conditions, a negative deviation of the dendritic E GABA from the somatic value (i.e., ⌬E GABA ) is a quantitative measure of the efficacy of Cl Ϫ extrusion (Jarolimek et al, 1999;Khirug et al, 2005). Somatic and dendritic E GABA values were determined from the current-voltage rela-tions, obtained by sequentially clamping the membrane potential at different holding potentials with a 5 mV increment and a 10 s step interval.…”