Proc. Nati. Acad. Sci. USA Vol. 89, pp. 241-245, January 1992 Physiology Patchy accumulation of apical Na' transporters allows cross talk between extracellular space and cell nucleus (Na' channel/gene activation/cell fusion) H. OBERLEITHNER*, S. WUNSCH, AND S. SCHNEIDER Department of Physiology, University of Wurzburg, Rontgenring 9, 8700 Wurzburg, Federal Republic of Germany Communicated by Gerhard Giebisch, September 16, 1991

ABSTRACT Intracellular Na' activities and local current distances between the sites of measurements of the Na' densities were measured in fused Madin-Darby canine kidney signal were determined. cells using Na' and voltage-sensing microelectrodes. Na' that Lectin Binding. The apical plasma membranes of fused enters the cell across the apical plasma membrane accumulates MDCK cells exhibit receptors for wheat germ agglutinin initially in the nudeoplasm, several seconds ahead of its (WGA), a lectin serving as a suitable marker to identify the appearance in the cell cytoplasm. The spatial distribution of Na' reabsorbing "principal type" cell of the renal collecting Na' currents, produced by a local superfusion of the cell duct (9). In contrast, single (nonfused) MDCK cells exhibit surface, indicates a nonuniform, patchy accumulation of apical receptors for peanut agglutinin (PNA), a lectin identifying the Na' transporters in the vicinity of the nucleus. Such pathways "intercalated type" cell of the collecting duct (10). In short, for direct Na' flux between extracellular space and cell nucleus cells were exposed for a few minutes to tetramethyl- could be potentially important for gene activation. rhodamine B isothiocyanate (TRITC)-labeled WGA and flu- orescein isothiocyanate (FITC)-labeled PNA (100 .tg/ml; In most somatic cells RNA synthesis is preceded by enlarge- Medac, Hamburg, F.R.G.) and after rinsing were exposed to ment of the nucleus and dispersion of the chromatin, indi- incident light of either 510-560 nm (TRITC fluorescence) or cating gene activation (1). Cation concentration in the cell 450-490 nm (FITC fluorescence) wavelength (Fig. 1). nucleus is crucial for the expression of puffs, the visible Intracelular Na+. Fabrication of the liquid ion-exchange correlate for activation of specific loci in chromosomes (2). microelectrodes and calibration procedures have been de- Na+ could be an important signal for such a mechanism scribed (11). In short, glass micropipettes were drawn from because it counterbalances net negative charges of acidic single-barreled glass tubings (1.5 o.d., with internal fiber; proteins accumulated in the nucleoplasm in significant Hilgenberg, Malsfeld, F.R.G.). The inner wall ofthe micropi- amounts (3). Some years ago, it was proposed that there is a pette tips were siliconized, baked at 200°C for 1 h, and then direct communication pathway for Na+ between extracellu- backfilled with Na+ resin (Na+ ionophore I, no. 71176; lar space and cell nucleus (4, 5). This conclusion was based Fluka). Electrodes had tip diameters of -1 ,um, an electrical the resistance of -1011 fQ, and a 90% response time of <1 s. The on the observation that 22Na injected systemically into voltage response ofthe microelectrodes induced by changing jugular vein ofrats reaches at the same time virtually identical Na+ activities in the range of 5-50 mM at constant ionic equilibrium concentrations in blood (i.e., extracellular space) background (140 mM K+/2.0 mM Mg2+/0.01 mM Ca2+) was and in nuclei of liver cells (5). virtually linear on a semilogarithmic scale. Cytoplasmic and nucleoplasmic free Na+ concentrations were evaluated ac- METHODS cording to the equations Nac = Nao exp(10)[(V~aVC)/S]- and Na. = Na0 exp(10)[(VNa - Vn)/S], respectively. VNa and We tested the hypothesis on a putative communication Vc are the Na+ electrochemical and the electrical potential pathway between extracellular space and cell nucleus with differences, respectively, measured in the cell cytoplasm. electrophysiological experiments in cultured Madin-Darby Vna and Vn are those measured in the nucleoplasm (Fig. 2a). canine kidney (MDCK) cells, an established cell line derived Na0 is the extracellular Na+ concentration and the symbol S from the collecting duct of dog kidney (6). Single epithelial is the electrode slope. The bathing solution was connected to cells were fused to "giant" cells according to published ground via a Ag/AgCl agar bridge that served as the reference techniques (7, 8). electrode. Measurements were performed with single- Cell Fusion. In short, cells were grown in culture and barreled Na+-sensitive and conventional microelectrodes, harvested by trypsin/EDTA treatment. Then cells were both inserted in the cell nucleus or in the cell cytoplasm. The fused to giant cells by a modified polyethylene glycol method neighboring electrode tips of the Na+-sensitive and the (7). The multikarya were maintained in culture for at least 3 conventional microelectrodes (in either nucleus or cyto- days until most nuclei were either fused or decomposed. plasm) were only a few micrometers apart from each other. Giant cells intermingled between single MDCK cells in a Although four cell penetrations were necessary for one subconfluent monolayer were studied on the stage of an individual measurement, we preferred the application of inverted microscope applying high-resolution interference single-barreled microelectrodes over double-barreled ones; contrast microscopy (Zeiss, IM 35; objective 63/1.4; oil). the manufacture of single-barreled microelectrodes is tech- Giant cells (diameter, 50-200 ,um) grown on glass were nically simple and the electrical properties of such electrodes superfused with modified Ringer's solution composed of are highly reproducible. 122.5 mM NaCl, 5.4 mM KCI, 1.2 mM CaC12, 0.8 mM MgCl2, On the other hand, giant MDCK cells can withstand 1.0 mM Na2HPO4, 10 mM Hepes, 5.5 mM glucose, and multiple impalements without compromising cell function. In titrated to pH 7.4. The experiments were videotaped and the Abbreviations: WGA, wheat germ agglutinin; PNA, peanut aggluti- The publication costs of this article were defrayed in part by page charge nin; TRITC, tetramethylrhodamine B isothiocyanate; FITC, fluo- payment. This article must therefore be hereby marked "advertisement" rescein isothiocyanate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

241 Downloaded by guest on October 2, 2021 242 Physiology: Oberleithner et al. Proc. Natl. Acad. Sci. USA 89 (1992) least -20 mV in Ringer's solution (see above) and in which the membrane potential remained constant over the course of an individual experiment were evaluated. The intracellular change in Na' concentration in nucleus and cytoplasm was induced by a step change of extracellular Na' from 26 to 126 mM (equivalent amounts of NaCl were substituted with mannitol). Local Superfusion Technique. The method is outlined in Fig. 3. First, the overall resistance (R) of the cell membrane was evaluated by the intracellular injection of short current pulses (AI; 1 nA; 0.5 s), while the resulting voltage deflections (AV) were measured with another microelectrode inserted into the cytoplasm (13). It must be pointed out that this experimental procedure presumes a uniformity of the cell membrane resistance that is inconsistent with our data shown below and thus represents a simplification. Then the mouth of a patch pipette (tip diameter, -4 gm) was placed in close proximity to the apical plasma membrane. The pipette was filled with modified Ringer's solution in which 100 mM Na' was replaced with choline (Na' concentration, 30 mM). While the cell was superfused systemically with regular Ringer's solution (Na' concentration, 130 mM) local pertur- bations (1 s duration) were performed at various sites of the apical cell membrane and the resulting changes ofthe plasma membrane potential (AV) were measured with an intracellu- lar microelectrode. It must be assumed that the magnitude of AV is influenced by the distance between the tip of the microelectrode and the site ofthe local superfusion due to the cable properties of the plasma membrane. Thus, the equation given in Fig. 3 (Lower) is only an approximation. Since we kept the AV-sensing microelectrode strictly in the cell periphery while moving the local superfu- sion stepwise from the cell center toward the edge of the cell (i.e., locating the superfusion site closer to the microelec- trode tip), we rather underestimated the steepness of the lateral AVgradients. However, this experimental error seems to be negligibly small because the AV pattern is virtually the same independent of whether the distance between the superfusion site and AV-sensing electrode remains constant or variable. An example for a measurement at constant distance is given in Fig. 4a. The local superfusion was performed by means of an electronically controlled home- built injection device. It is based on pneumatic valves that can be precisely opened for 1-s periods to allow a preset pressure to be maintained on the pipette-injection system. FIG. 1. Fluorescence micrographs of an epithelial MDCK mono- The transient local reduction of Na+ concentration (i.e., layer composed of a fused giant cell (center) surrounded by individ- change of the electrochemical potential for Na+, AENa+) ual single cells (periphery). The apical plasma surface was double- the a transient flow of labeled with two fluorescent lectins, WGA (TRITC fluorescence) and hyperpolarized membrane, indicating PNA (FITC fluorescence). (a) Only the fused MDCK cell (center) Na+ current (INa+). From the resulting voltage changes (AV) shows clear WGA staining (TRITC fluorescence) ofthe apical plasma and the specific membrane resistance (R), we estimated INa+ membrane, whereas the neighboring single cells (periphery) virtually at various loci of the apical plasma membrane. We video- lack WGA receptors. (b) Most of the single cells show PNA staining taped the experiments by means of a video-image analysis (FITC fluorescence of the apical plasma membrane, whereas the system (Java, Jandel, Corte Madera, CA) and thus could giant cell (center) lacks PNA receptors. determine the individual locus, the superfused area (cm2), and then could relate it to local INa+. Due to the different a few experiments, we applied four microelectrodes simul- refraction index of the systemic (130 Na+) and the local (30 taneously (one Na'-sensitive and one conventional micro- Na+) superfusion solution, the locally superfused apical electrode inserted in the cell nucleus and another pair of surface area could be identified and analyzed from the electrodes inserted in the cell cytoplasm). Although techni- videotape. In Fig. 4a, the dark area below the number 18 is cally feasible, we found that subsequent (repetitive) super- caused by local superfusion with the low Na+ concentration. fusion measurements with only one pair of microelectrodes inserted in either cell nucleus or cell cytoplasm are more reliable because, on the one hand, repetitive (and timely RESULTS exact) changes of extracellular Na' concentration in the Fig. 1 shows a MDCK monolayer with a fused MDCK cell in superfusate are highly reproducible and not prone to any its center. The apical plasma membranes were double-labeled experimental artefacts, whereas, on the other hand, the with two fluorescent lectins, WGA (Fig. la) and PNA (Fig. electrical response (e.g., response time of the Na' electrode) lb). The fused MDCK cell clearly binds WGA, whereas most of two different pairs of microelectrodes may differ slightly, of the single MDCK cells, surrounding the fused cell, lack thus causing unpredictable artefacts. Only experiments in WGA receptors but bind PNA. According to studies in intact which the plasma membrane potential difference (VT) was at collecting duct, epithelium cells with PNA receptors were Downloaded by guest on October 2, 2021 Physiology: Oberleithner et al. Proc. Natl. Acad. Sci. USA 89 (1992) 243

Cytoplasm Nucleus 7- ucleus Na vc vVc v Na Vn E I Eu .-I z Cytoplasm

1T-.Z;er-isa aZ- { -

1 48 pm 1 4 6 8 10 12 14 a b Time (s)

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0 Z 2- u 0 / x2/2t z *C .I 2 >1 .- I DNa 2.5.10-6cm2s. |- EIuc 0) on 0 10 20 30 40 50 60 70 Ctn12 n =12 C Distance from nucleus (pm) d Cytoplasm Nucleus FIG. 2. (a) Measurement of the Na' signal in nucleus and cytoplasm of giant MDCK cells with Na'-selective and conventional microelectrodes. VcNa and Vc are the Na' electrochemical and the electrical potential differences, respectively, measured in the cell cytoplasm. VnI and V,, are those measured in the nucleoplasm. (b) When giant MDCK cells (n = 12) were superfused with low Na+ (26 mM) Ringer's solution, free Na' concentration in nucleus and cytoplasm averaged 5.4 ± 0.8 and 4.0 ± 1.0 mM, respectively. The rapid substitution of extracellular low Na+ Ringer's with regular Ringer's solution (126 mM Na') led to an increase of intracellular free Na' concentration in nucleus and cytoplasm. The change of cytoplasmic Na' concentration was 4.5 s delayed compared to the Na' change in the nucleoplasm. The two recording sites were 48 jum apart (see a). (c) Relationship between the arrival time of the Na' signal in the cytoplasm and the distance of the cytoplasmic recording site from the nucleus. The curve was calculated according to the modified Einstein equation (D = x2/2t) using an estimate of the diffusion coefficient for Na+ in the cytoplasm (DNa; ref. 12). In the equation t is the diffusion time of cytosolic Na+ for a given distance x. (d) Na+ concentration changes induced by a sudden increase of extracellular Na+ from 26 to 126 mM were measured in the cell nucleus and-with a time delay-in the bulk phase of the cytoplasm of 12 giant MDCK cells (± SEM).

termed intercalated-type cells (10), while cells with WGA in nucleus and cytoplasm in response to altered extracellular receptors were assumed to resemble principal-type cells of Na+. the renal collecting duct (9). We have chosen the giant (WGA Surprisingly, the Na+ signal arrived first in the cell nucleus positive) MDCK cell because its unique geometry (7) allows and subsequently, with some delay, in the cytoplasm (Fig. the precise evaluation of the intracellular Na+ pathway of a 2b). The delay time was a function of the distance between principal-type cell in culture. Giant cells, attached to the glass the nucleus and the site in the cytoplasm where the Na+ surface with their basolateral cell membranes and grown in a signal was measured (Fig. 2c). This indicates that Na+ enters subconfluent monolayer, were superfused with Ringer's so- the nucleus first and then diffuses into the bulk phase of the lution and punctured with conventional and Na+-sensitive cytoplasm. Na+ diffusion through the nuclear envelope is microelectrodes (Fig. 2a). First, the cell nucleus was pene- most likely mediated by the nuclear pores that represent a trated with the two microelectrodes and the change of the highly permeable pathway for ions (14). Applying the mod- intracellular Na+ concentration, the so-called Na+ signal, ified Einstein equation (D = x2/2t) and using an estimate of was measured in response to a rapid concentration step of the diffusion coefficient D for Na+ in the cytoplasm (D extracellular Na+ from 26 to 126 mM. Then the electrode tips 2.5.10-6 cm2/s; ref. 12), the diffusion time t ofNa+ for a given were placed in the cytoplasm of the same cell at a given distance x in the cytosolic phase can be calculated. It turns distance from the nucleus and the superfusion procedure was out that -3 s is needed for Na+ to diffuse over a distance of repeated. Note that over the course of this experiment the 40 Am. This is in reasonable agreement with our data (Fig. 2c) conventional microelectrode-its tip inserted either in the and consistent with the view that the delay ofthe cytoplasmic nucleoplasm or in the cytoplasm-recorded the electrical Na+ signal is due to the diffusion of Na+ from the nucleo- potential difference that was electronically subtracted from plasm into the peripheral cytoplasm. The initial slopes of the the individual Na+ electrochemical potential difference mea- Na+ concentration changes in nucleus and-after the respec- sured by the Na+-sensitive microelectrode. This procedure tive time delay-in cytoplasm, induced by the rapid increase allowed the continuous monitoring offree Na+ concentration of extracellular Na+, were estimated from the individual Downloaded by guest on October 2, 2021 244 Physiology: Oberleithner et al. Proc. Natl. Acad. Sci. USA 89 (1992)

c-4 30A R E U 25- Plasma Membrane 2V0- TAV Na1 z 15-

'Na R 10- 5- FIG. 3. Local superfusion technique. (Upper) Overview. (Lower) Details. R, overall plasma membrane resistance; AV, voltage change 0-inal In=n215iu nz10 n10 Mr:71 ofthe plasma membrane; AENa+, change of the Na+ electrochemical 0-10 11-20 21-30 31-40 41-50 51-60 potential difference across the superfused plasma membrane patch; b Distance from nucleus (pm) INa, local Na+ current (for further details see Methods). FIG. 4. (a) Giant MDCK cell in culture. White Numbers corre- tracings as shown in Fig. 2a. It turned out that the initial spond to local Na' currents (INa; ,uA/cm2) at various sites of the apical plasma membrane. They were obtained in patches of 225 ALm2 change of Na+ concentration was 3-fold higher in the by the local superfusion technique. Asterisk marks position of the nucleus than in the bulk phase of the cytoplasm (Fig. 2d). It AV-sensing microelecrode during the measurement. Please note that, indicates that there is a rapid communication pathway be- in this experiment, the distance between superfusion site and cell tween extracellular space and cell nucleus. Obviously, Na+ center (nucleus) varies, while the distance between superfusion site passes through the nucleoplasm before it diffuses into the and AV-sensing microelectrode remains virtually constant. (b) Local bulk phase of the cytoplasm. Na' currents (n = number of observations; mean + SEM) obtained In previous studies, it was suggested that the endoplasmic from different sites of the apical plasma membrane are shown in dependence of the distance from the cell nucleus. reticulum could serve as a functional channel network to span the distance between plasma membrane and nucleus (4, 5). the supranuclear apical surface (i.e., 5% of the total apical An alternative explanation for the observed phenomenon surface) ':24% of total cell INa can be recovered. Obviously, could be a nonuniform distribution ofNa+ transporters in the Na' channels are being inserted into the apical plasma apical plasma membrane. To test this hypothesis, a method membrane in the vicinity of the cell nucleus. Based on the was developed to measure local densities of Na+ currents steepness ofthe lateral gradients in Na' channel density that (INa+) at various sites ofthe apical plasma membrane ofgiant are similar to the ones observed in skeletal muscle (15), it can MDCK cells grown in a subconfluent monolayer. Fig. 3 be assumed that the lateral mobility of the Na+ transporters introduces the local superfusion technique. Local changes of is restricted and that are anchored in the Na+ concentration in individual areas of the apical plasma they supranuclear membrane induce local INa+ that can be monitored by micro- surface of the apical plasma membrane. Another view is that electrodes. In Fig. 4a one example is given. It is obvious that the endoplasmic reticulum and, in particular, the Golgi ap- the density of local INa (,.A/cm2) measured at various loci of paratus are sandwiched between the nucleus and the supra- the apical plasma membrane is high near the nucleus and nuclear portion of the apical membrane. Then a high density small in the cell periphery. The data are summarized in Fig. of appearance of channels in the supranuclear area and a 4b. They clearly show a nonuniform distribution of local INa, lower one in more peripheral areas could be expected. indicating that the majority of apical Na+ transporters is accumulated in the direct vicinity of the cell nucleus. In DISCUSSION addition, we measured in seven giant cells the total apical cell surface (4553 ± 311 Mm2) and the apical area just above the The supranuclear accumulation ofthe Na' transporters in the nucleus (240 ± 29 Mm2). Furthermore, we evaluated total cell apical plasma membrane explains the phenomenon that an INa of the apical plasma membrane obtained from the total extracellular Na' signal arrives first in the cell nucleus if the membrane resistance and the cell membrane voltage changes latter is located close to the apical membrane. The major induced by a Na+ concentration step of the systemic super- portion of Na+ enters the cell across the apical membrane fusion solution. From these data it was calculated that from right above the nucleus, passes the nucleoplasm, and then Downloaded by guest on October 2, 2021 Physiology: Oberleithner et al. Proc. Natl. Acad. Sci. USA 89 (1992) 245

Lumen Blood cleus via the apical plasma membrane is extruded from the intracellular compartment by the basolateral Na'-K' pump in exchange for K+. Thus, the nonuniform distribution of apical Na' transporters could serve as a mechanism for nuclear K+ accumulation. However, we want to emphasize that inferences based on these fused, giant MDCK cells plated on glass coverslips may not necessarily apply to renal epithelia. Since the activation of genes depends on the actual Na' and K+ concentration in the nucleoplasm (4, 5, 21-23), a rapid and direct cross talk between extracellular space and nucleus could be important to control basic processes such as gene activation, cell growth, and cell differentiation. We thank Dr. Stefan Silbernagl for encouraging support during the course of the experiments, Dr. Luis Reuss for critical reading of a previous version ofthe manuscript, Mrs. Barbara Schuricht and Mrs. Birgit Gassner for excellent technical assistance, Mrs. Margit Schulze for preparing the figures, and Mrs. Sabine Kopp for pre- paring the manuscript. This work was supported by Deutsche Forschungsgemeinschaft SFB 176-A6. 1. Ashburner, M. (1971) Nature New Biol. 230, 222-223. 2. Kroeger, H. (1963) Nature (London) 200, 1234-1235. FIG. 5. Hypothetical model of the transcellular Na' pathway in 3. Krohne, G. & Franke, W. W. (1980) Exp. Cell Res. 129, a renal epithelial cell and its potential role in gene activation. Caused 167-189. by the supranuclear accumulation of Na' transporters in the apical 4. Siebert, G. & Langendorf, H. (1970) Naturwissenschaften 57, plasma membrane, Na' is imported directly into the cell nucleus. 119-124. Na' extrusion across the basolateral cell membrane occurs via the 5. Langendorf, H., Siebert, G. & Nitz-Litzow, D. (1964) Nature Na'-K+-ATPase that exchanges Na' for K+. This leads to the (London) 204, 888. accumulation of K+ in the nucleoplasm facilitated by the intranuclear 6. Valentich, J. D. (1981) Ann. N. Y. Acad. Sci. 372, 384-405. negative potential difference. This model is based on the assumption 7. Kersting, U., Joha, H., Steigner, W., Gassner, B., Gstraun- that the cell nucleus is sandwiched between the apical and basolateral thaler, G., Pfaller, W. & Oberleithner, H. (1989) J. Membr. plasma membranes, whereas the bulk phase of the cytoplasm is Biol. 111, 37-48. located in the (lateral) cell periphery. It strongly supports the ion 8. Oberleithner, H., Schmidt, B. & Dietl, P. (1986) Proc. Natl. hypothesis of gene regulation (2, 21). Acad. Sci. USA 83, 3547-3551. 9. Minuth, W. W., Gilbert, P., Rudolph, U. & Spielman, W. S. diffuses further into the bulk phase ofthe cytoplasm. In other (1989) Histochemistry 93, 19-25. words, the cell nucleus serves as a permeable intracellular 10. O'Neil, R. G. & Hayhurst, R. A. (1985) Am. J. Physiol. 248, compartment linking the apical plasma membrane loaded 449-453. with Na' transporters to the bulk phase of the cytoplasm. 11. Steiner, R. A., Oehme, M., Ammann, D. & Simon, W. (1979) Anal. Chem. 51, 351-353. The pores of the nuclear envelope are considered to be too 12. Horowitz, S. B. & Fenichel, I. R. (1970) J. Cell Biol. 47, large to act as barriers against ions (14, 16, 17). In murine 120-131. pronuclei (18) and in nuclei of salivary gland cells of Dro- 13. Oberleithner, H., Kersting, U. & Hunter, M. (1988) Proc. Natl. sophila larvae (14, 19), negative intranuclear potential differ- Acad. Sci. USA 85, 8345-8349. ences were detected. In nuclei of giant MDCK cells, we 14. Paine, P. L., Moore, L. C. & Horowitz, S. B. (1975) Nature measured (in reference to the cytoplasm) an intranuclear (London) 254, 109-114. W. J. potential of -4.1 0.9 mV (n = 12; mean SEM). It is most 15. Almers, W. R., Stanfield, P. R. & Stuhmer, (1983) Physiol. (London) 336, 261-284. likely explained by a Gibbs-Donnan potential due to the high 16. Dingwall, C. & Lakey, R. A. (1986) Annu. Rev. Cell Biol. 2, concentration of nuclear acidic proteins (3) and/or due to 367-390. negative electrical changes of the phosphate backbone of the 17. Feldherr, C. M. & Akin, D. (1990) J. Cell Biol. 111, 1-8. DNA. Such a negative intranuclear potential could be of 18. Mazzanti, M., DeFelice, L. J., Cohen, J. & Malter, H. (1990) physiological relevance: karyophilic proteins enter the nu- Nature (London) 343, 764-767. cleus through the nuclear pores, allowing entry only to 19. Loewenstein, W. R. & Canno, Y. (1962) Nature (London) 195, polypeptides equipped with the correct nuclear signal se- 462-464. 20. Finlay, D. R., Newmeyer, D. D., Hartl, P. M., Horecka, J. & quence (20). In the larval salivary glands of Chironomus Forbes, J. (1989) J. Cell Sci. Suppl. 11, 225-242. nuclear K+ activity increased by a factor of 2.6 as dormant 21. Wuhrmann, P., Ineichen, H., Riesen-Willi, U. & Lezzi, M. larvae developed into prepupae (21). This puzzling finding (1979) Proc. Natl. Acad. Sci. USA 76, 806-808. could be due at least in part to the negative intranuclear 22. Leake, R. E., Trench, M. E. & Barry, J. M. (1972) Exp. Cell potential that facilitates nucleoplasmic K+ accumulation. It is Res. 71, 17-26. tempting to hypothesize (Fig. 5) that Na' entering the nu- 23. Kroeger, H. (1966) Exp. Cell Res. 41, 64-80. Downloaded by guest on October 2, 2021