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. T IP elongation in fungal hyphae is an excellent example of polarized growth; new plasma membrane and cell wall are deposited almost exclusively at the apex. Evidence collected over the past two decades has shown that most, if not all, tip-growing organisms generate transcellular electric currents at their apices (1, 3, 7, 20, 23, 38, 41, 42); in many cases the appearance of localized inward current precedes the emergence of a nascent tip and accurately predicts its site (20, 22, 42). Such findings suggest that electric currents play a causal role in polarized growth, but the underlying mechanism(s) is/are unclear. One possibility is that the electric field created by the current flow induces both cytoplasmic and membrane asymmetry by redistributing charged macromolecules and organelles (16). However, it has proved difficult to measure reliably the internal field owing to its small magnitude and the possibility of introducing artifacts during electrode impalement. Previous findings from our laboratory (using an extracellular vibrating probe) showed that growing hyphae of the water mold Achlya generate a transcellular electric current as depicted in Fig. 1 (20). Protons carry current into the tip and we postulated that they cross the plasma membrane by symport (co-transport) with amino acids, particularly methionine (8, 19). The current entering the tip flows through the hyphal cytoplasm toward the region of outward current. We suggested that the outward current was driven by proton extrusion from an electrogenic H+-ATPase (19). To complete the current loop, charge flows through the extracellular medium from trunk to tip. This proton circulation may be expected to make the cytoplasm at the tip acidic and electropositive with respect to the zone of outward current. This concept of the nature and genesis of the transcellular current has now been reinforced by the use of intracellular microelectrodes. Three aspects of the model were examined in this study: (a) the existence of a primary ion pump in the plasma membrane, (b) the electrogenicity of methionine transport, and (c) the presence of a cytoplasmic voltage gradient near the apex. The most striking finding was the presence of an intracellular electric field of 0.2 V/cm immediately behind the hyphal tip. The intracellular electric field was abolished under conditions that block the flow of transcellular current, indicating that these two phenomena are closely linked in the physiology of this organism. Although the function of the field remains to be determined, calculations show that it is of sufficient magnitude to transport anionic cellular constituents to, and localize them at, the growing tip by selfelectrophoresis. (c> The Rockefeller University Press, 0021-9525/86/04/I 209/08 $1.00 The Journal of Cell Biology, Volume 102, April 1986 1209-1216 1209 on July 1, 2017 jcb.rress.org D ow nladed fom