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 (a[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 a[Cl-](i). When the pipette solution contained 150 mM Cs+ ([Cs+](pipette)), a[Cl-](i) increased to a value higher than the passive a[Cl-](i). An increase of a[Cl-](i) with the [Cs+](pipette) was not due to the simple blockade of net KCC by the intracellular Cs 1 since a[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 a[Cl-](i) with the [Cs+](pipette) was reduced by 1 mM furosemide, but not by 20 mu M bumetanide or Na+-free external solution, indicating that the accumulation of [Cl-](i) in the [Cs 1]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 a[Cl-](i). On the other hand, Rb+ mimicked K+ in the pipette solution. These results indicate that K+ and Rb 1, but not Cs 1,Li 1,or Na 1, can act as substrates of KCC in LSO neurons.