Biochemical Studies of Synapses in Vitro. II. Potassium Transport

Previous studies of rat brain synaptosomes in vitro demonstrated modulation of protein synthesis by ionic constituents and endogenous energy availability, and suggested that isolated nerve endings may support active ion transport. Utilizing a rapid filtration technique to measure intrasynaptosomal potassium accumulation, the various parameters influencing uptake of cold potassium and ⁴²K were studied at 23°. Immediately after isolation, synaptosomes contained an average of 0.092 μmole of potassium/mg of protein. At 23° with 100 mM sodium and 10 mM potassium in the external medium, the synaptosomal potassium increased to approximately 0.14-0.25 μmole/mg of protein after 8 min. The optimal potassium accumulation occurred with 50 mM sodium present in the medium. With 50 mM sodium, potassium content rose to 0.20-0.30 μmole/mg of protein after 8 min. At this optimal level of sodium (50 mM), potassium concentrations greater than 10 mM produced minimal increase in potassium accumulation. Neither adenosine triphosphate, adenosine diphosphate, glucose, increased oxygen concentration, nor the substrates α-ketoglutarate, succinate, fumarate, and glutamate influenced potassium accumulation. Ouabain was an effective inhibitor only of the sodium-dependent potassium accumulation. In sodium-free medium ouabain had essentially no effect on potassium ion accumulation. In the presence of 50 mM sodium and ouabain (10^-4 M), the total potassium content of the synaptosomes declined by 54%. The extent of inhibition by ouabain was equivalent to the extent of activation by sodium. Both 2,4-dinitrophenol and potassium cyanide inhibited potassium accumulation. Total potassium and ⁴²K uptake by synaptosome decreased as a function of time after isolation with the most marked reduction in the ouabain-inhibitable component. Total synaptosomal volume was measured with the use of N-methyl-[¹⁴C]antipyrene, and the potassium content per milligram of protein was expressed in synaptosomal potassium concentrations. With 10 mM potassium and 50 mM sodium, there was an increase of intrasynaptosomal potassium concentration from approximately 71 mM following isolation to 103 mM, representing a significant accumulation of potassium against a concentration gradient. The present studies demonstrate that isolated rat brain synaptosomes support active potassium transport.