Reversibility and Cation Selectivity of the K+-Cl−Cotransport in Rat Central Neurons

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Abstract
The reversibility and cation selectivity of the K+-Cl− cotransporter (KCC), which normally extrudes Cl− out of neurons, was investigated in dissociated lateral superior olive neurons of rats using the gramicidin perforated patch technique. Intracellular Cl− activity (α[Cl−]i) was maintained well below electrochemical equilibrium as determined from the extracellular Cl− activity and the holding potential, where the pipette and external solutions contained 150 mM K+([K+]pipette) and 5 mM K+([K+]o), respectively. Extracellular application of 1 mM furosemide or elevated [K+]o increased α[Cl−]i. When the pipette solution contained 150 mM Cs+([Cs+]pipette), α[Cl−]i increased to a value higher than the passive α[Cl−]i. An increase of α[Cl−]i with the [Cs+]pipette was not due to the simple blockade of net KCC by the intracellular Cs+ since α[Cl−]i, with the pipette solution containing 75 mM Cs+ and 75 mM K+, reached a value between those obtained using the [K+]pipette and the [Cs+]pipette. The higher-than-passive α[Cl−]i with the [Cs+]pipette was reduced by 1 mM furosemide, but not by 20 μM bumetanide or Na+-free external solution, indicating that the accumulation of [Cl−]i in the [Cs+]pipette was mediated by a KCC operating in a reversed mode rather than by Na+-dependent, bumetanide-sensitive mechanisms. Replacement of K+ in the pipette solution with either Li+ or Na+mimicked the effect of Cs+ on α[Cl−]i. On the other hand, Rb+ mimicked K+ in the pipette solution. These results indicate that K+ and Rb+, but not Cs+, Li+, or Na+, can act as substrates of KCC in LSO neurons.