Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion‐selective microelectrodes.

Abstract

1. Intracellular Cl- activity (aCli) and membrane potential (Em) were measured in frog dosal root ganglion neurones (DRG neurones) using double-barrelled Cl--selective microelectrodes. In standard Ringer solution buffered HEPES (5 mM), equilbrated with air or 100% O2, the resting membrane potential was -57.7 .+-. 1.0 mV and aCli was 23.6 .+-. 1.0 mM (n = 53). The value of aCli was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl- led to reversible fall in aCli. Initial rates of decay and recovery of aCli were 4.1 and 3.3 mM min-1, respectively. During the recovery of aCli when following regurn to standard Ringer solution, most of the movement of Cl- occurred against the driving force for a passive distribution. Changes in aCli when external Cl- was removed too high to be accounted for by electrodiffusion. 3. The intracellular accumulation of Cl- was dependent on the extracellular Cl- activity (aClo). The relationship between aClo and aClo had a sigmoidal shape with half-maximal activation of about 50 mM-external Cl-. 4. The steady-stateaCli depended on the simultaneous presence of extracellular Na+ and K+. Similarity, the active reaccumulation of Cl- after intracellular Cl- depletion was abolished in the absence of either Na+ or K+in the bathing solution. 5. The reaccumulation of Cl- was inhibited by furosemide (0.5 .times. 10-3 M) or bumetandine (10-5 M). The decrease of aCli observed in Cl--free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aCli. Cells were estimated to shrink in Cl- free solutions to about 75% their inital volume, at an initial rate of 6% min-1. 7. The present results provide direct evidence for the active accumulation of Cl- in DRG neurones. The mechansism of Cl- transport is electrically silent, dependent on the simultaneous presence of external Cl-, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+: K+: Cl- co-transport system mediates the active transport of Cl- across the cell membrane of DRG neurones.