Reversibility and cation selectivity of the K⁺-Cl^- cotransport in rat central neurons

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.