Ion Fluxes in Dialyzed Squid Axons

A method will be described which allows the control of internal solute composition in squid axons while they are engaging in metabolically dependent ion transport. A glass tube, about 250 m m long and 0.175 m m in diameter containing a central 20 m m long region of porous glass, was inserted into the axon, and the porous region was positioned in the middle of the axon. When solutions of a defined composition were driven through the capillary at rates of from 0.6 to 1.2 #l/rain, measurements showed that Na + equilibrated with the axoplasm surrounding the porous glass with a time constant of 3-5 rain. Other measurements showed that substances such as ATP, phenol red, water, Na +, and I crossed the porous glass readily, whereas molecules such as hemoglobin, aldolase, and myokinase had time constants for transfer of the order of 103-105 rain. In use, therefore, the porous glass capillary behaves as a dialysis membrane in that it allows small molecules to pass readily but retains protein. Internal dialysis fluids with a variety of compositions were used for the experiments performed, but most commonly the dialysis fluid had the following composition: K isethionate, 151 re_M; K aspartate, 151 mM; NaC1, 80 mM; taurine, 275 raM; KaEDTA, 0.1 mM; MgCI~, 0.1-2.0 rnM. These constituents were selected for the following reasons. Taurine and K isethionate concentrations closely approximate those normally present in axoplasm, whereas the NaC1 concentration was selected to be closer to that of axons that had been isolated for some time. The concentration of K aspartate was made equal to the sum of the aspartate -b glutamate concentrations in axoplasm; MgC12 was added as an activator of membrane ATPase. The EDTA was an abnormal ingredient; its addition rendered nontoxic any heavy metals that might have been introduced into internal dialysis fluids as impurities in glassware, reagents, or isotopes. As possible sources of metabolic energy for ion movements across the membrane, a variety of high-energy phosphate compounds were used. The principal compound used was ATP at concentrations in dialysis fluid of 25-10,000/~M. An equimolar coneentration of MgC12 was added to such ATP solutions, since ATP strongly chelates Mg ++. Phosphoarginine was frequently added to dialysis fluids at concentrations of 0.5-10.0 mM; a mixture of ATP and phosphoarginine is designated "fuel" in the results to be presented. Under conditions where an experiment required a very low [ATP], 2 rnM K C N was added to dialysis fluid; isotopes such as ~Na were added when Na efflux was to be measured. In most experiments, dialysis fluids were not buffered, mainly because it was not easy to decide on substances suitable for this function. Instead, before use, fluids were carefully adjusted to p H 7.0 with small amounts of K O H or HC1. In some more recent experiments, K TES (tris [hy-