uasi-intact axon of the crash

Transcription

1 176 Proc. Japan Acad., 75, Ser. B (1999) [Vol. 75(B), A voltage clamp method with internal perfusion for q uasi-intact axon of the crash By Tetsuji HIRONAKA and Yoshio IKARI Department of Pharmacology, Teikyo University School of Medicine, Kaga, Itabashi-ku, Tokyo (Communicated by Masanori OTSUKA, M.J.A., June 15, 1999) Abstract: A voltage-clamp system with internal perfusion for quasi-intact crayfish giant axons was developed using a partition method where three pools were formed by two partitions. The membrane potential was extracellularly recorded across the one side partition whose resistance was highly increased using a water-absorbing material, Sephadex G25 mixed with vaseline and liquid paraffin so that no short circuiting correction became necessary. Axon membranes were voltage-clamped at the center by feeding current through the other side pool. For the internal perfusion with the axoplasm left intact, the membranes of the lateral pools were perforated with a 0.1% of saponin-treatment for min, through which the axon was intracellularly "perfused" with a reasonable diffusion rate. This system was well bearable to long-term experiments under both extracellularly and intracellularly controlled conditions of the cell. axon; Key words: Partition method; voltage clamp; crayfish. internal perfusion; saponin-perforated; quasi-intact Introduction. Recently, conventional patch clamp method is widely used to investigate membrane properties of a variety of cells. However, there exist a number of disadvantages in the method. (1) Patch electrode glass composition affects ion channel currents.2~'5~ (2) A great amount of tension is given to the membrane to create a giga seal at the tip of glass-capillary so that an artificial conductance might be produced. In fact, Bosma (1989) reported a giant conductance for chloride channel recorded with the patch clamp method. In addition, leakage channels are physiologically activated by changes in cell volume. ~"2)"4>"5~'2 (3) A patch dissociates cytoplasm which may link with a function of excitable membrane.' "6> It will be necessary to confirm the inherent properties of channels en masse for the results obtained by the patch clamp technique. Before the development of the patch clamp technique, a potentiometric method was devised and improved for voltage-clamp techniques and has offered a great advantage especially for the experiments on nerve fibers of small sizes4)'24~ and muscle fibers,10~"7 and even for giant axons." "3~ However, all these methods used a feedback amplifier for the membrane stability opposing the flow of injury current and have not successfully measured absolute resting potentials. The present study was intended to develop a simple system in which voltage clamp and internal perfusion with the membrane and cytoplasm kept intact can be applied. For these purposes, the potentiometric and saponin-perforated internal perfusion techniques were utilized in crayfish giant axons. The special aim of this paper is to establish a system to study resting membrane behaviors in an intact condition. Materials and methods. Preparation. The dissecting method is the same as described in the preceding paper.8~ In short, the giant axons of the circumesophageal connectives of the crayfish, Procambarus clarkii, were isolated, and a single medial giant axon was desheathed and cleared of more than 99 % of adjoining tissues under a binocular microscope. The dissected axon was mounted in an acrylic chamber as schematically drawn in Fig. 1A. In principle, the partition method is identical to that described in the preceding papers,8~ but in practice it is slightly different. To accelerate the speed of voltage clamp, the width of the partitions and pools were made as narrow as possible (Fig. 1A). The chamber has three pools (L-side pool, R-side pool, and center pool, C) with two partitions, each of which consisted of three subpartitions (I,M,O). Typical dimensions of the chamber are (um): central pool, 300; inner subpartition, ;

2 No. 6] Potentiometric voltage clamp in the crayfish axon 177 Table I. Axon constants of crayfish giant axons determined at 10 C Fig. 1. Schematic diagrams of principle of the method. A: Chamber and experimental arrangement. Stippled areas (I, M, 0) represent subdivisions of each partition. The partitions and axon are roughly to scale. L, C and R are left, central and right pools. Vm, membrane potential. Ir, recorded current. B: Equivalent circuit of the current pathway. Current injected through the left pool flows along the arrows. The heads of two or three letters, I and R show current and resistance. The second small letter, m, i, e and 1, membranous, internal, external, and left-external. The last M, L and R, stand for membrane, left and right respectively. middle subpartition, 200; outer subpartition, 250; right lateral pool, 480; and left lateral poo1,17,000 with a volume of 0.5 ml. The inner and outer subpartitions constitute a mold of a mixture consisting of 30-35% vaseline (Iwai Chemical Co., Ltd., Tokyo, Japan) and 65-70% liquid paraffin (Iwai Chemical Co., Ltd., Tokyo, Japan). The middle subpartition (M) was specialized for a water-absorbing layer consisting of 20-25% Sephadex G25 Superfine (Pharmacia, Fine Chemicals, Uppsala, Sweden) added to the mixture of 10% vaseline and 90% liquid paraffin. The liquid paraffin was used to reduce viscosity of vaseline so that its mixture can encroach on narrow spaces at the immediate vicinity of the axon surface. The membrane potential was recorded with two external electrodes filled with 3M KCI, one is at the right lateral pool and the other is at the central pool (Fig. 1A). A current-feeding electrode was set at the left pool. Both membranes of the lateral pools were depolarized with the offset solution8 when they were not perforated for internal perfusion. Solutions and chemicals. The standard bathing saline was a modification of Van Harreveld's (1936) solution. containing (mm): NaC1205, KCl 5.4, MgC12 2.6, CaC1213.5, HEPES 3 with ph 7.5. Internal solution contained (mm): NaCI 15, K-methanesulfonate 144.7, K3-citrate 37.5, HEPES 3, titrated to ph 7.4 with KOH. The chemicals used are 4-aminopyridine (4-AP, Tokyo Kasei Kogyo Co., Ltd, Tokyo, Japan), tetrodotoxin (TTX, Sankyo Co., Ltd., Tokyo, Japan) and saponin (Sigma Chemical, St. Louis, U. S. A.). All experiments were performed at a temperature of 10 C. Results. Axon constants of crayfish giant axons. Axon constants were determined using a specially designed chamber for these accurate measurements. The dimensions of the chamber were 500 pm in width at the center, measuring membrane resistance and are 1,750 pm in length of the partition, measuring axoplasm resistance. The membrane resistance in control and depolarized axon were measured at the central pools using the voltage clamp technique. For measurements of the resistivity of the axoplasm, two microelectrodes were inserted across the long partition, through which a constant current was injected and the resistance was calculated from the potential drop. The results are summarized in Table I. Equivalent electric circuit of the system current. A system analysis was carried out in Fig. lb where the

3 178 T. HIRONAKA and Y. IKARI [Vol. 75(B), external electrode at the right-pool is inside as one of the variations of the system application and current is fed through the left pool. The advantage of this variation is that RmR in Fig. lb can participate in the external resistance (ReR) to be constructed. The current is assumed to flow as illustrated with arrows and the electric resistance of each pass-way is designated as in Fig. 1B. The membrane current Im is obtained as a fraction of the Ir recorded at the center which consists of Im, Ii, and Ie, being equal to the injected current. Thus, Im=Ir-II-Ie, [1] Ii = Im + Ie. [2] Assuming that each current Ix is proportional to a reciprocal of each resistance Rx in Fig. 1, the Im is expressed as, Im = Ir {ReL(RiR + RmR + ReR)/ [(RmL + RIL + ReL + R) (RiR + RmR + ReR + RmM)]} where 1/R =1/RmM + 1/(RiR + RmR + ReR). [3] The short-circuiting factor for the potentiometric is, Ve/Vm = (ReR + RmR)/(ReR + RmR + RiR), [4] where Ve is the membrane potential recorded with the extracellular electrodes. If ReR and ReL are infinitely large, R = RmM and eq. [4] reduces to unity, giving Ve = Vm. Eq. [3] also reduces to Im = Ir. For more than 99% accuracy in the potential measurement, the barrier resistance (ReR) to be constructed at the right-side (Fig. 1A) requires a value more than 2.06 MS1, in which the axon constants in Table I and the dimensions described in Materials and methods were used with the axon diameter of 200 pm. As a result, this variation in the system contributes by about 9% to the barrier resistance. It should be noted that the relevant membrane resistance (RmM) is not involved in the short circuiting factor. Therefore, even if the insulation be not good enough, the true membrane potential can be obtained from the factor with the condition that the residual membrane potential at the lateral depolarized membrane is equal to zero.8 Current family recorded with the potentiometric voltage clamp. The membrane potential was monitored with the two external electrodes and the current was injected through the left pool as shown in Fig. 1. In Fig. 2A, the current family did not show any notch, suggesting a good space clamp. To facilitate the space clamp, both ends of the axon were made electrically blind, resulting in a reflex of the bifurcated current (Ie in Fig. 1B). Fig. 2B shows the current family in 1 mm 4-AP. The tail currents were considerably decreased with slow outward currents remained which might correspond Fig. 2. Sodium and potassium currents of the crayfish giant axon under the potentiometric voltage clamp. Resting potential, -86 mv. Holding potential, -95 mv. A, control. B,1 mm 4-AP. to the TEA-insensitive K currents.22~ Outward peak currents which have fast rising phases are clearly seen in both current families. Series resistance of the crayfish giant axon. In Fig. 3, the peak sodium current-voltage relations of an axon in control and TTX are shown where no series resistance compensation was carried out. The relations did not show a noticeable voltage shift with TTX. This result is well reconciled with the fact that TTX ( nm) selectively reduces the maximum sodium conductance without changing the kinetics of the remaining sodium currents or without changing voltage dependence of the peak sodium conductance and of the steady-state inactivation.3~'7~"8 '23~ Warashina and Fujita (1983) also described in the crayfish axon that the dis-

4 No. 6] Potentiometric voltage clamp in the crayfish axon 179 Fig. 4. Internal perfusion through the saponin-perforated lateral pools. The perforated lateral pools were immersed with the internal solution initially and it was replaced with Van Harreveld's solution at the arrow. A rate of diffusion of the solution was monitored by observing membrane potential at the center (Fig. 1A). The time constant was 7.2 min. Fig. 3. Current-voltage relations of the crayfish giant axon in control and TTX. Resting potential, -82 mv. Holding potential, -95 mv. Symbols refer to peak currents. Filled circle, control. Filled square, filled triangle and filled rhombus are 10, 15 and 30 min after 1 nm of TTX respectively. tortion of the current profile caused by the series resistance was not serious when checked under the condition of a reduced Na-current density in the presence of 2 nm TTX. On the other hand, Hassen and Lieberman (1988) electrically measured the series resistance of the crayfish axon, giving an average value of 6 S cm2 ranging from 1 to 30 S cm2. If this were the case in Fig. 3 where the series resistance was not compensated, TTX should have produced a noticeable shift along the voltage axis in the peak sodium current-voltage relations, since the change in current by TTX amounted to nearly 2 malcm2 at most (Fig. 3). It remains to be confirmed whether or not the special efforts made in the preparation to obtain a good insulation (Materials and methods) might have removed some diffusion barrier which might be involved in the previous measurements of the series resistance. In this connection, it is interesting to note that the accumulated external K+ concentration in the periaxonal space during outward K+ current returns to normal in an exponential fashion, with a time constant of -2 msec, which is about 25 times faster than the case in squid axons.21~ Internal perfusion. For the internal perfusion, both membranes of the lateral pools were treated with 0.1% saponin contained in the internal solution for min to perforate the membranes,19~ through which an internal perfusate was supplied with an appropriate hydrostatic pressure. Fig. 4 shows a reasonable stability of the membrane after the treatment and a perfusion condition which was monitored by observing a change in membrane potential when the internal solution was replaced with Van Harreveld's solution at the lateral pools. The time constant determined by fitting the following equation was 7.2 min. Vm = Vstart - (Vstart - Vsteady) [ 1- exp (-t/'t) ]. Longevity of the preparations. The axons in this system with the internal perfusion survived well for a long-term experiments, say about 3 to 4 hours. So far we have not obtained data for more than 4 hours simply because we stopped the experiments. Discussion. The principle of the voltage-clamp method shown in Fig. 1 is primarily based on the improvement of the insulating technique of the partition constructed on the surface of the axon membrane, which simplified its system by making the feedback amplifiers unnecessary to avoid additional current injection to the preparation and made it possible to measure the membrane potential without insertion of a microelectrode. Thus, the longevity of the preparations was attained. Further, removal of chloride ions from the offset solution in the lateral reference pool also largely contributed to the longevity of the preparation.8 Internal "perfusion" was carried out with the axoplasm left intact through saponin-perforated holes where a large molecule of substances can be applied.l9~

5 180 T. HIRO N AK Aa nd Y. IKARI [Vol. 75(B), These three factors altogether have made it possible to do experiments of a cell under an analytical control with an intact condition. In this voltage-clamp system, a time resolution sounds enough from the clearly seen outward peak currents in Fig. 2. References 1) Bosma, M. M. (1989) J. Physiol. 410, ) Costa, G., and Armstrong, C. M. (1987) Biophys. J. 51, 49a (Abstr.). 3) Cuervo, L. A., and Adelman, W. J., Jr. (1970) J. Gen. Physiol. 55, ) Dodge, F. A., and Frankenhaeuser, B. (1958) J. Physiol. 143, ) Furman, R. E., and Tanaka, J. C. (1988) Biophys. J. 53, ) Hassan, S., and Lieberman, E. M. (1988) Neuroscience 25, ) Hille, B. (1968) J. Gen. Physiol. 51, ) Hironaka, T., and Ikari, Y. (1999) Proc. Japan Acad. 75B, ) Hoffmann, E. K., and Dunham, P. B. (1995) Int. Rev. Cytol. 161, ) Ildefonse, M., and Rougier, 0. (1972) J. Physiol. 222, ) Julian, F. J., Moore, J. M., and Goldman, D. E. (1962) J. Gen. Physiol. 45, ) Lang, F., Busch, G. L., Ritter, M., Volki, H., Waldegger, S., Gulbins, E., and Haussinger, D. (1998) Physiol. Rev. 78, ) Lund, A. E., and Narahashi, T. (1981) Neurobiol. (Little Rock) 2, ) McCarty, N. A., and O'Neil, R. G. (1992) Physiol. Rev. 72, ) Macknight, A. D. C., and Leaf, A. (1977) Physiol. Rev. 57, ) Matsumoto, G., and Sakai, H. (1979) J. Membrane Biol. 50, ) Moore, L. E. (1972) J. Gen. Physiol. 60, ) Narahashi, T., Moore, J. W., and Scott, W. R.(1964) J. Gen. Physiol. 47, ) Ohtsuki, I., Manzi, R. M., Palade, G. E., and Jamieson, J. D. (1978) Biol. Celluolaire 31, ) Sarkadi, B., and Parker, J. C. (1991) Biochim. Biophys. Acta 1071, ) Shrager, P., Starkus, J. C., Lo, M.-V. C., and Peracchia, C. (1983) J. Gen. Physiol. 82, ) Soria, B., and Rojas, E. (1986) Gen. Physiol. Biophys. 6, ) Takata, M., Moore, J. W., Kao, C. Y., and Fuhrman, F. A. (1966) J. Gen. Physiol. 49, ) Tasaki, I., and Bak, A. F. (1958) J. Neurophysiol. 21, ) Warashina, A., and Fujita, S. (1983) J. Gen. Physiol. 81,