Amiloride-Sensitive Sodium Flux and Potentials in Perfused Carcinus Gill: Preparations

Sodium and chloride fluxes, as well as transbranchial potentials (TBP) were studied in isolated perfused gill filaments of the crab Carcinus mediterraneus. Experiments were carried out in media that were either hyposmotic to the perfusion solution (asymmetrical conditions) or isosmotic (symmetrical conditions). Fluxes were found to be diffusional in gills under asymmetrical conditions; amiloride induced an inhibitory effect on influxes, without affecting TBP. Under symmetrical conditions, TBP was −7.6±2.3mV, suggesting that the electrogenic ion pump contributes significantly to the development of TBP. Immediately after addition of 2.5 × 10−4 moll−1 amiloride to the external solution, sodium influxes were reduced to 31% of those in the control group, and TBP was significantly hyperpolarized from −7.6 to −14.8 mV. The absence of Ca2+ under symmetrical conditions diminished TBP hyperpolarization. Half-maximal inhibition of sodium influxes by amiloride was at 7 × 10−5 moll−1. This low amiloride affinity is typical of low resistance leaky epithelia. Sodium transport is discussed as an amiloride-affected influx, probably as a Na/H antiport.

Electrophysiological properties of Achlya hyphae: ionic currents studied by intracellular potential recording.

The electrical properties of the water mold Achlya bisexualis were investigated using intracellular microelectrodes. Hyphae growing in a defined medium maintained a membrane potential (Vm) of -150 to -170 mV, interior negative. Under the conditions used here, this potential was insensitive to changes in the inorganic ion composition of the medium. Changes in external pH did affect Vm, but only outside the physiological pH range. By contrast, the addition of respiratory inhibitors caused a rapid depolarization without affecting the conductance of the plasma membrane. Taken together these findings strongly suggest that the membrane potential is governed by an electrogenic ion pump rather than by an ionic diffusion potential. Previous work from this laboratory showed that Achlya hyphae generate a transcellular proton current that enters the growing tip, flows along the hyphal length, and exits distally from the trunk. These initial experiments used an extracellular vibrating electrode, and I now report intracellular electrical recordings which support the hypothesis that protons enter the tip by symport with amino acids and are expelled distally by a proton-translocating ATPase. Most significantly, current flowing intracellularly along the hyphal length is associated with a cytoplasmic electric field of 0.2 V/cm or greater. Conditions that inhibit the current also abolish the internal field, suggesting that these two phenomena are closely linked.

Origin of the membrane potential in plasmodial droplets of Physarum polycephalum. Evidence for an electrogenic pump.

Spherical droplets, derived from Physarum plasmodia by incubation in 10 mM caffeine, seemed to be an excellent system for electrophysiological studies because they were large (less than or equal to 300 micrometer in diameter) and because they tolerated intracellular electrodes filled with 3 M KCl and 10 mM EDTA for a few hours. Intact plasmodia, by contrast, gave valid records for only a few minutes. Under standard conditions ([K+]o = 1 mM, [Na+]o = 5 mM, [Ca++]0 = 0.5 mM, [Mg++]o = 2 mM, and [Cl-]o = 6 mM at pH 7.0), the potential difference across droplet membranes was -80 to -120mV, interior negative. The membrane potential was only slightly sensitive to concentration changes for the above-mentioned ions, and was far negative to the equilibrium diffusion potentials calculated from the known internal contents of K, Na, Ca, Mg, and CL (29.4, 1.6, 3.7, 6.5, and 27.8 mmol/kg, respectively). Variations of external pH did have a strong influence on the membrane potential, yielding a slope of 59 mV/pH between pH 6.5 and 5.5. In this pH range, however, the equilibrium potential for H+ (assuming 6.2 less than or equal to pHi less than or equal to 7.0) was greater than 75 mV positive to the observed membrane potential. Membrane potential was directly responsive to metabolic events, being lowered by potassium cyanide, and by cooling from 25 to 12 degrees C. This ensemble of results strongly indicates that the major component of membrane potential in plasmodial droplets of Physarum is generated by an electrogenic ion pump, probably one extruding H+ ions.