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 (alpha[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 alpha[Cl(-)](i). When the pipette solution contained 150 mM Cs(+) ([Cs(+)](pipette)), alpha[Cl(-)](i) increased to a value higher than the passive alpha[Cl(-)](i). An increase of alpha[Cl(-)](i) with the [Cs(+)](pipette) was not due to the simple blockade of net KCC by the intracellular Cs(+) since alpha[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 alpha[Cl(-)](i) with the [Cs(+)](pipette) was reduced by 1 mM furosemide, but not by 20 microM 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 alpha[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.
Kakazu
Y
Y
Cellular and System Physiology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
Uchida
S
S
Nakagawa
T
T
Akaike
N
N
Nabekura
J
J
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D005260:Female; D005665:Furosemide / Q000494:pharmacology; D008297:Male; D009474:Neurons / Q000378:metabolism*; D009847:Olivary Nucleus / Q000166:cytology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D027981:Symporters; D000096922:K Cl- Cotransporters
D002034:Bumetanide; D002352:Carrier Proteins; D002412:Cations; D002586:Cesium; D002712:Chlorides; D004232:Diuretics; D005665:Furosemide; D011188:Potassium; D027981:Symporters; D000096922:K Cl- Cotransporters