Ammonium action on post‐synaptic inhibition in crayfish neurones: implications for the mechanism of chloride extrusion
Authors: Aickin C. Claire , Deisz R. A. , Lux H. D. 
Published: December 2014

1. The reversal potential of the Cl−‐dependent, inhibitory post‐synaptic potential (Ei.p.s.p.) was measured in the isolated crayfish stretch receptor neurone using two intracellular micro‐electrodes. The difference between Ei.p.s.p. and the resting membrane potential (Em), the i.p.s.p. driving force, was reversibly decreased by addition of NH3/NH4+, and the mechanism of this decrease was investigated.

2. The NH3/NH4+‐induced decrease in i.p.s.p. driving force was dose‐dependent with an onset at about 0·2 mM. Ei.p.s.p. always remained more negative than Em or, when the neurone was spontaneously firing, the threshold potential. Em and resting membrane resistance (Rm) also decreased in a dose‐dependent fashion. Synaptic conductance (gs) increased with low doses, but decreased on application of 20 mM‐NH3/NH4+. All the effects were fully reversible on return to normal Ringer solution.

3. Intracellular acidification (substitution of 50% Cl− by acetate compared with isethionate) considerably reduced the i.p.s.p. driving force. Simultaneous application of NH3/NH4+ and acetate‐substituted Ringer solution caused a similar decrease in the driving force to application of the same concentration of NH3/NH4+ under normal conditions. Increasing the extracellular pH at which a given concentration of NH3/NH4+ was applied caused a smaller decline in the i.p.s.p. driving force. These results suggest that intracellular acidification decreases the i.p.s.p. driving force and that the NH3/NH4+‐induced decline is caused by an action of the ammonium ion.

4. Elevation of extracellular K+ (K+0) decreased the i.p.s.p. driving force, Em and Rm, and increased gs. Reduction of K+0 had the converse effects on all parameters.

5. Application of Rb+ or Cs+ mimicked the effects of NH3/NH4+. Substitution of K+0 by Rb+, Cs+ or NH3/NH4+ opposed or even reversed the increase in i.p.s.p. driving force when Na+ was used as the substitute. The effectiveness of the various cations in decreasing the driving force was in the following order: Rb+ > NH4+ > K+ > Cs+.

6. Inhibition of the Na pump by ouabain or K+‐free Ringer solution caused a gradual reduction in the i.p.s.p. driving force. Since the driving force also decreased when the Na+ gradient probably was increased (elevated K+0), this suggests a dependence on the K+ gradient rather than the Na+ gradient or the Na pump itself.

7. Frusemide (6 × 10−4 M) reversibly decreased the i.p.s.p. driving force and Em, and increased gs. Rm was not significantly affected. Application of frusemide in the presence of 5 mM‐Rb+ and vice versa, caused a further reduction in the driving force. The recovery of the driving force on removal of either agent was slowed by the presence of the other.

8. Application of 4,4‐diisothiocyanostilbene‐2,2‐disulphonic acid (DIDS; 10−4 M) caused spontaneous firing and reduced Ei.p.s.p. to the threshold potential. Rm and gs increased. The effects were slowly reversible on removal of the drug.

9. It is proposed that the i.p.s.p. driving force is maintained by a K+—Cl− co‐transport mechanism, driven by the K+ gradient. The K+ site exhibits the binding selectivity: Rb+ > NH4+ > K+ > Cs+ and the mechanism is inhibited partially by frusemide and completely by DIDS.