Effect of NH4+/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop.

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K+. In excised membrane patches the luminal K+ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (Vm) in slow-whole-cell (SWC) recordings and the open probability (Po) of K+ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of Vm can be recorded. The pipette solution contained K+ 125 mmol/l and Cl- 32 mmol/l. Intracellular pH was determined by 2',7'bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH4+/NH3 to the bath. In the presence of NH4+/NH3 intracellular pH acidified by 0.53 +/- 0.11 units (n = 7). Inhibition of the Na+2Cl-K+ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62 +/- 0.14 units (n = 7). In SWC experiments Vm of TAL cells was -72 +/- 1 mV (n = 70). NH4+/NH3 depolarised Vm by 22 +/- 2 mV (n = 25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH4+ from 24 +/- 3 mV to 7 +/- 3 mV. Under control conditions the single-channel conductance of TAL K+ channels in CAN experiments was 66 +/- 5 pS and the reversal voltage for K+ currents was 70 +/- 2 mV (n = 35). The Po of K+ channels in CAN patches was reduced by NH4+/NH3 from 0.45 +/- 0.15 to 0.09 +/- 0.07 (n = 7). NH4+/NH3 exposure depolarised the zero current voltage of the permeabilised patches by -9.7 +/- 3.6 mV (n = 5). The results show that TAL K+ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH4+/NH3 exposure at concentrations which are physiologically relevant because Na+2Cl-K+(NH4+) cotransporter-mediated import of NH4+ exceeds the rate of NH3 diffusion into the TAL. K+ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH4+ uptake occurs by the Na+2Cl-K+ cotransporter. The findings that, in the presence of NH4+/NH3 and furosemide, Vm is not completely repolarised and that K+ channels are not activated suggest that the respective K+ channels may in addition to their pH regulation be inhibited directly by NH4+/NH3.