J. Exp. Biol. (1966), 45, 251-267 251 With 10 text-figures Printed in Great Britain MEMBRANE POTENTIALS IN AMOEBA PROTEUS BY M. S. BINGLEY R.A.F. Institute of Aviation Medicine, Farnborough, Hants. {Received 16 February 1966) INTRODUCTION The possibility that the initiation of pseudopods together with the direction of cytoplasmic streaming may be induced by local depolarization of the membrane has been advanced from time to time for many years past. But is it difficult to find a direct statement to this effect in the literature. Amici (1818) suggested an electrical theory to account for streaming in plant cells and more recently Kitching (1961) considers the possibility of depolarization of the cell surface initiating contraction. When he discussed Hahnert's work (Hahnert, 1932) on the response of amoebae to electricity he pointed out that movement of the pseudopods towards the cathode may be an enhancement of 'local currents associated with local excitation'. It is one thing to put forward a hypothesis but another to obtain convincing measurements which are free from the suspicion of doubt and artifact. Membrane potentials were first seriously measured in Amoeba proteus by Telkes in 1931. Using large electrodes, she obtained low values of membrane potential but produced extremely valuable information on various depolarizing agents such as potassium and sodium chloride. Buchtal & Peterfi (1936) obtained low values of potential for A. proteus. Wolfson (1943) produced convincing values for membrane potential in Chaos chaos and, using electrodes of large diameter, he obtained potentials as high as workers using more modern micro- electrodes. More recently Riddle (1962) working on PeUomyxa caroHnensis recorded values of — 90 mV. for the membrane potential and carried out convincing studies on the effect of increasing concentrations of external electrolytes on the membrane potential. Observations were made on the lack of effect of certain metabolic inhibi- tors on membrane potential. However, all these workers appear to have regarded the cell as being uniform in its electrical properties over its whole surface and to have dis- regarded the possibility that in a freely streaming amoeba, complete with actively advancing pseudopods showing well-defined hyaline caps, the membrane properties may vary topographically and that this might be reflected in differences of membrane potentials. It must be realized that the advent of micromanipulators capable of inserting a microelectrode into the tip of a fast-moving pseudopod is only relatively recent. Bingley & Thompson (1962), using such a manipulator, were able to test directly certain hypotheses on the possible connexion between cytoplasmic streaming, pseudopod formation and electrical potential changes. They carried out a series of experiments involving direct potential measurement with saline-filled glass micro- electrodes and found that the potential in the pseudopod was some 30 mV. less than the rear region, which on average was of the order of — 65 mV. A great deal of sub- sidiary evidence pointed to the conclusion that cytoplasmic streaming was related to 252 M. S. BlNGLEY these potential differences. It is of note that these cells were freely streaming and un- constrained in any way. Japanese work, notably that of Kishimoto (1957), indicated a possible relationship between streaming and changes of membrane potential in the slime moulds. Kamiya (1964) and Tasaki & Kamiya (1964) later attempted to con- firm the potential difference reported by Bingley & Thompson (1962) and failed to do so, Tasaki being very much concerned with the presence of a circulating current postulated by Bingley & Thompson. Barueva (1965), working on freely moving A. proteus, was able to confirm the presence of a low membrane potential in the pseudo- pod but explained these observations in terms of short-circuiting of the membrane potential through improper sealing of the membrane round the microelectrode in the region of the pseudopod. Two problems face modern workers investigating membrane potentials in fresh- water A. proteus. The first is the simple fact that these cells live in fresh water and not in saline media, and the second arises from the almost universal use of hyperfine microelectrodes. Incidentally botanists face the same problems, which are complicated by the toughness of plant cell walls. Recently Bingley (19646, 1965 a) investigated the behaviour of hyperfine glass microelectrodes in solutions ranging from dilute Chalk- lev's solution on the one hand, which is one-hundredth the molarity of Ringer, to 3*0 M potassium chloride on the other. He came to the conclusion that electrode characteristics such as tip potential and electrode resistance tend to be exaggerated in very dilute solutions and are a function of external ion concentration. Changes in these could well mask bio-electric potentials encountered on passing through the mem- brane of fresh-water A. proteus. However, the author has never used hyperfine micro- electrodes for recording membrane potential in fresh-water amoebae and has always treated his electrodes to reduce resistance and tip potential by widening their tips before insertion (Bingley, 1964a, b, 1965 a). It is also noticeable that earlier workers, notably Telkes (1931), Wolfson (1943) and Riddle (1962), obtained consistent poten- tials using saline-filled microelectrodes of large diameter (100 fi) on large cells. Bingley (19656) repeated the experiments of Bingley & Thompson (1962) and presented the results in the form of photographic records, laying a great stress on the absence of tip potential and constant base-line. However, all this evidence is not as yet absolutely convincing since we have as yet no concrete proof that when low potentials are en- countered in the pseudopod the microelectrode has in reality penetrated the membrane and is not simply indenting the membrane as in the experiments on sea-urchin eggs (Hiramoto, 1959). This paper is mainly concerned with experiments designed to test the validity of the hypothesis that the low potential recorded from an advancing pseudopod is a genuine membrane potential. In the process other observations have been made and recorded which are relevant to the process of amoeboid locomotion. Particular attention has been paid to membrane potentials recorded in the rear region of streaming amoebae and to observations made on changes of electrode res- ponse while recording these potentials. Previous work on the behaviour of electrodes not inserted into the cell (Bingley, 19646, 1965 a) has enabled possible interpretations of the state of ions within A. proteus to be put forward. Membrane potentials in amoeba proteus 253 METHODS Cultures of A. proteus were grown in Chalkley's medium. They were fed on Tetra- hymena pyriformis in mass culture four times a week. The composition of Chalkley's m i medium is: 1-37 HIM NaCl, 0-027 mM KC1, 0-047 ^ NaHCO3, 0-007 mM Naj,HPO4, mM 0-007 CaHPO4. All reagents used were of analytical quality. Glass-distilled water was used throughout. The experimental technique for recording membrane potentials has already been described (Bingley, 1964a). Source of current pulse Membrane potential recorded here Cathode follower '(to oscilloscope) voltage trace Current Voltage electrode electrode \ Cathode follower current trace Current recording (to oscilloscope) Fig. 1. The experimental technique involved in measuring the alternating voltage and current across the amoeba membrane. The current electrode is shown inserted into the amoeba. This carries the inset waveform, which is expanded to show its nature. The voltage electrode is inserted into the cell to record the membrane potential in that particular region. Superimposed on this is an alternating voltage. This.is shown from the output on the cathode follower on the voltage trace. Current passing in the system is measured by means of a second cathode follower measuring voltage across a x-o Mfl resistance, R.I. This is passed through a capacitor, C, to remove the d.c. component and a typical waveform inset is shown. Essentially, amoebae were introduced into an experimental chamber, open at the top, containing dilute Chalkley's solution. The amoebae attached themselves to the bottom surface of this chamber where they streamed freely. Membrane potentials were recorded by means of saline-filled glass microelectrodes (3-0 M-KCl) which were manipulated with Leitz micromanipulators. These were connected to a high input impedance, low grid current, cathode-follower system. This device has been catho- dally screened (Donaldson, 1958) and will record a square wave of 1 kc across a 10 MXl resistor with very little distortion. Potentials were recorded by means of a camera 254 M- S. BlNGLEY attached to an oscilloscope whose time-base was driven by a rotating potentiometer. By this means it was possible to produce linear sweeps as slow as i cycle in 5 min. Electrode resistance was continuously monitored throughout the experiments, in- volving single microelectrode recording by means of pulses fed at 5/sec, or 1 sec. intervals through a known value of resistance to the microelectrode via a capacitor. Since the internal conductivity of amoeba cytoplasm is as yet not accurately known the increase of resistance recorded by the electrode after insertion through the mem- brane cannot be ascribed to any particular component in the system. Electrode resis- tance recorded when the electrode is out of the cell will be referred to as R.E. Double microelectrode penetration Two