Efflux of Chloride Ions during the Action Potential of Nitella

Abstract

THIS communication is prompted by a report of Spyropoulos, Tasaki and Haywood¹ that they were unable to confirm the findings of Gaffey and Mullins² that in Chara there is an efflux of chloride ions during an action potential. It would appear, however, that the experimental conditions of Spyropoulos et al. were not such as to make a demonstration possible. This is shown by the following calculations. A Nitella cell, 0.06 cm in diameter by 4 cm in length, has a volume of 0.011 cm³ and very nearly 0.66 cm² of surface area. The volume of the cell is nearly all sap and if its [Cl] is taken as 100 µmole/cm³ then a cell contains 1.1 µmole Cl⁻. With a membrane capacity of 1 µF/cm² and an action potential of 100 mV, 1 × 10⁻¹² mole of Cl⁻ must be lost to produce the potential change, per cm² or 0.66 pmole for the Nitella cell. The cells used by Spyropoulos et al.. had 1,000 c.p.m. chlorine-36 and 1 µmole Cl⁻, so that each c.p.m. represented 10⁻⁹ mole Cl⁻ and the expected loss for an action potential is about 1/1,000th of a c.p.m. Forty stimuli were delivered—this would increase the efflux but the efflux was collected in 20 fractions so that the total efflux to be expected for the experiment would be 2/1,000ths of a c.p.m. in each fraction. In the experiments with Chara² the quantities of Cl⁻ lost were 500–5,000 times greater than that calculated above and were equivalent to K⁺ loss, while the action potential was 200 mV, or considerably greater than that in Nitella. The K⁺ loss demonstrated by Spyropoulos et al. ought to equal Cl⁻ loss, but in the absence of information on the specific activity of their potassium-42 in the cell no calculation is possible. The specific activities of potassium-42 and chlorine-36 as supplied by the Atomic Energy Commission, Oak Ridge, are as 2.000 : 1, and for equal loading times the intracellular potassium would be preferentially labelled. What does seem clear is that the extent to which cells were labelled with chlorine-36 was inadequate to detect an extra efflux of activity (over the resting Cl⁻ efflux and over background).