ATP-driven, Na(+) -independent inward Cl(-) pumping in neuroblastoma cells.

摘要

In immature neurones, the steady-state intracellular Cl(-) concentration [Cl(-) ] (i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+) -K(+) -2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl(-) ] (i) around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, [Cl(-) ] (i) decreased quickly (t(1/2) < 5 min), suggesting a high Cl(-) permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl(-) uptake was strongly inhibited (60-65%) while steady-state [Cl(-) ] (i) decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP](i) and [Cl(-) ] (i) by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl(-), in parallel with ATP recovery. Internal bicarbonate did not affect Cl(-) pumping, suggesting that Cl(-) /HCO(3) (-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36) Cl(-) uptake nor steady-state [Cl(-) ] (i) were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl(-) pumping in N2a cells.