Membrane potentials in pinched-off presynaptic nerve ternimals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials.
1. Some physiological properties of tissue fractions from rat brain homogenates have been examined. Of the three fractions studied (presynaptic nerve terminals, mitochondria and fragmented membranes), only the nerve terminals (synaptosomes) have the ability to accumulate 42K from physiological salt solutions. 2. The ability to accumulate and retain K is lost if synaptosomes are exposed to very hypotonic solutions. The K uptake and total K content is reduced by ouabain and by inhibitors of glycolysis and oxidative phosphorylation. 3. These results suggest that synaptosomes in physiological saline accumulate K against a concentration gradient, and may have K diffusion potentials across their surface membranes. The voltage-sensitive fluorescent probe, 3,3'-dipentyl 2,2'-oxacarbocyanine (CC5), was used to test this possibility. 4. In the squid axon, the fluorescent emission of CC5 is directly proportional to membrane potential; depolarization causes an increase in fluorescence. 5. The fluorescence of synaptosomes ('synaptosome fluorescence') treated with CC5 is increased when [K]o is increased or [K]o is reduced; replacement of external Na by Li or choline has little effect on the synaptosome fluorescence. In quantitative terms, synaptosome fluorescence is proportional to log ([K]o plus 0-05[Na]o). Rb is about as effective as K in enhancing synaptosome fluorescence; Cs is about 1/4 as effective. The effect of increased [K]o is reversible. 6. The fluorescence data provide corroborative evidence that there is normally a large K gradient ([K]o smaller than [I]i) across the synaptosome surface membrane. The data suggest the [K]i may be in excess of 100 mM. 7. Replacement of Cl- by methylsulphate did not significantly affect the relationship between synaptosome fluorescence and [K]o, nor did removal of external Ca. 8. The fluorescence of CC5-treated mitochondria, membrane fragmnets, or lysed synaptosomes is unaffected by changes in the K concentration of the medium. 9. Veratridine and gramicidin D, both of which enhance Na permeability (PNa) in some intact tissues, increase synaptosome fluorescence when added to the standard medium. The increment is greatly reduced or abolished when external Na is replaced by choline. 10. If synaptosomes are first Na-loaded (by pre-treatment with cyanide + iodoacetate), and then placed in a choline medium, addition of gramicidin D significantly decreases fluorescence. This effect could be explained if, with [Na]o smaller than [Na]i, the increase in PNa causes the synaptosomes to hyperpolarize. 11. The veratridine-induced increase in synaptosome fluorescence was prevented by 3 times 10- minus 7M tetrodotoxin, which also blocks the depolarizing effect of veratridine in intact neurones. 12. The main conclusion is that synaptosomes may retain resting membrane potentials and the ability to increase Na permeability.