[K]O. [Cl]o. This phenomenon is attributed to a delayed equilibration by. viscosity.

Transcription

1 J. Physiol. (1971), 215, pp With 11 text-figures Printed in Great Britain THE EFFECT OF POTASSIUM AND CHLORIDE IONS ON THE VOLUME AND MEMBRANE POTENTIAL OF SINGLE BARNACLE MUSCLE CELLS By BERT A. MOBLEY AND ERNEST PAGE From the Departments of Physiology and Medicine, The Pritzker School of Medicine, University of Chicago, Chicago, Illinois, U.S.A. (Received 5 November 1970) SUMMARY 1. In single barnacle skeletal muscle cells cell diameter has been measured as a function of external osmolality, and cell diameter and membrane potential have been measured during changes of external K and C1 concentrations ([K]0 and [Cl].) like those described in frog muscle by Hodgkin & Horowicz (1959). The diameter was monitored microscopically with a precision of % (S.D.). [K]o was varied from 1 to 18 mm, a range of concentrations which does not cause contracture. 2. At ph 8*0 the Cl permeability was so low that net KCl and water movements were absent. Such net movements were present at ph 4-5, corresponding to a change in the ratio (Cl conductance/k conductance) from approximately 1/12 at ph 8-0 to 1/2 at ph Characteristically long time constants were observed for membrane potential responses to a change in [K]0 and/or [Cl]o, even at constant [K]O. [Cl]o. This phenomenon is attributed to a delayed equilibration by diffusion within the system of sarcolemmal invaginations and T-tubules. The delay in the response was increased by introducing polyvinylpyrrolidone (PVP) into this system, presumably because PVP raises intratubular viscosity. 4. At ph 4*5 anomalous rectification for net movements of K was demonstrated by measurements of cell diameter and ofmembrane potential. INTRODUCTION In 1959 Hodgkin & Horowicz described the membrane potential transients produced by net movements of KCl across the cell membrane of single frog skeletal muscle cells. These transients were initiated by varying the external K or C1 concentrations, thereby disturbing the equili-

2 50 B. A. MOBLEY AND E. PAGE brium distributions of K and C1 defined by the classical study of Boyle & Conway (1941). Hodgkin & Horowicz predicted that the net movements of KC1 underlying the changes in membrane potential during the transition between equilibrium distributions must be accompanied by net movements of water and therefore by changes in cell volume. In the present study we have measured the time constants of such transient changes in volume directly in single barnacle skeletal muscle cells using a technique capable of detecting very small changes in cell diameter. Changes in the diameter of single muscle cells have been previously measured by other investigators in preparations from frog, crayfish, lobster and crab (Reuben, Lopez, Brandt & Grundfest, 1963; Reuben, Girardier & Grundfest, 1964; Gainer & Grundfest, 1968; Hays, Lang & Gainer, 1968). However, because the muscle fibres used were small, the techniques available to these investigators were relatively insensitive; it was therefore necessary to have recourse to large deviations from physiological osmolality or ionic composition and to elicit large deviations from the physiological volume of the fibre. Because the technique used in this paper was exceptionally sensitive, it was possible to examine very small net movements of KC1 in the physiological range of external K concentrations. Measurements of the membrane potential in parallel with the diameter measurements were used to confirm the results of the diameter measurement. In addition, we used the introduction of PVP into the system of sarcolemmal invaginations and T-tubules to increase the viscosity within this system and thereby to distinguish the contributions of the membranes lining this system to the depolarization produced by potassium ion. In this way it was possible to show how the membrane which lines the T-tubules and sarcolemmal invaginations of barnacle muscle affects the response of the membrane potential to K and C1 ions. In addition, both the diameter and membrane potential measurements suggest that anomalous rectification is present. Portions of this work have previously appeared in abstract (Mobley & Page, 1970). METHODS The depressor scutorum rostralis muscles of barnacles (Balanu8 nubilu8) were isolated by the method of Hoyle & Smyth (1963b). A small piece of shell with from one to four attached cells was then cut free from the shell and the rest of the muscle bundle. The cells were used in three types of experiments in solutions of variable osmolality and ionic composition: (a) measurement of the changes in cell diameter, (b) measurement of the resting membrane potential, and (c) measurement of the cellular contents of water and ions and of the intracellular ionic concentrations.

3 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 51 Measurement of the cell diameter Fig. 1 is a block diagram of the experimental assembly used for simultaneous monitoring of the cell diameter, resting membrane potential, temperature, and resting tension. The muscle fibre was suspended at 1-25 slack length in a lucite chamber. The chamber was mounted on the horizontal stage of a microscope equipped with a screw micrometer eyepiece. The fibre was mounted so that the major diameter of the cross-section was the diameter measured. The over-all magnification was x 40. The standard deviation of the diameter measurement (as directly determined on a muscle fibre) was ± 3 Iu, or % of the fibre diameters encountered ( ,#). Solution Microscope inlet with micrometer eyepiece Fig. 1. Diagram of the experimental assembly used for simultaneous monitoring of the cell diameter, resting membrane potential, resting tension and the temperature of the solution. After the fibre was placed in control sea-water in the chamber, the cell diameter was repeatedly measured until it became constant within the error of the measurement. A period of min was required to establish constancy. The test solution was then introduced and the diameter was measured at appropriate intervals until it again approached constancy. If the fibre remained in good condition during the test, it was returned to the control sea-water and the reversibility of the changes in cell diameter was examined; alternatively, a second test solution was introduced and further measurements were made. The length of the fibre remained constant during all measurements of the diameter. Changes in cell volume therefore appeared as changes in diameter. A tag of connective tissue or some other well-defined feature of the cell surface served as a point of reference for determining any changes in the length of any part of the fibre.

4 52 B. A. MOBLEY AND E. PAGE Since the present experiments involved much smaller deviations from isosmolality and much smaller net movements than those reported by previous workers (Huxley, Page & Wilkie, 1963; Girardier, Reuben, Brandt & Grundfest, 1963; Freygang, 1965), changes in the volume of the extracellular channels and T-tubules of the barnacle cell in response to changes in osmolality and ionic composition of the solution bathing the cell were considered to be negligible. The measurement of cell diameter was an extremely sensitive indicator of external osmolality. It proved impossible to weigh out reagents with a precision sufficient to assure complete isosmolality in modified sea waters of different chemical composition. Small differences in osmolality were therefore accepted, measured to within + 1 m- osmole (S.D.), and taken into account in interpreting the observed diameter changes. Measurement of the membrane potential For measurement of the resting membrane potential, fibres were mounted in the apparatus shown in Fig. 1. The membrane potential was measured between an intracellular micro-electrode and a grounded electrode in the external solution. The electrodes were filled with 3 m-kcl or with 3 m-kcl-4 % agar, respectively. The micro-electrodes had resistances of 3-7 MD and tip potentials < 6 mv. The extracellular electrodes had wide tips and resistances of kq. Before impalement, fibres stretched to 1-25 slack length were allowed to equilibrate in the control sea-water as for the measurement of cell diameter. The resistance and tip potential of the microelectrode were measured, the tip potential was compensated with the zero adjustment of the electrometer, and the cell was impaled. Next, the test solution was introduced for as long as required by the experiment, after which the fibre were re-perfused with the control sea water. The microelectrode was then withdrawn from the cell and its resistance and tip potential were again measured. Cells accepted for this study had membrane potentials of -70 to -80 mv in control sea water and maintained a constant tension. Measurement of cellular ion and water contents and of intracellular ionic concentrations To measure the intracellular contents of ions and water and the intracellular ionic concentrations, the tendon of each cell was tied with a thread, and the fibres and their attached shell were rinsed in a large volume of non-radioactive test solution. The fibres and shell were then transferred to a test-tube containing 30 ml. of test solution labelled with [14C]sucrose. The sucrose served as an extracellular tracer for solution in the sarcolemmal folds and T-system (which have been shown to contain significant Na (Brinley, 1968) and Cl (Gayton & Hinke, 1968)), and also for solution on the cell surface. The threads around the tendinous ends of the fibres were draped over the lips of the tubes and taped to the table. When thus suspended, the fibres were slightly stretched. The solution was stirred by a magnetic spinbar. Cells were incubated at C for periods of 45 min. The fibres and shell were then removed from the test-tube and placed in a dry dish. Each fibre was quickly severed near its insertion in the shell and drained against the side of the dish. The tendon was cut off, and the fibre was placed in a sealed, tared weighing bottle. Wet weight, dry weight corrected for the weights of salt and sucrose in the sucrose space, radioactivity due to [14C]sucrose, and the contents of K and Cl were determined by techniques previously described (Page, 1962; Page & Page, 1968).

5 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 53 Solution The artificial sea water which served as the control solution for this study was similar to that of Hoyle & Smyth (1963a); the buffering system was that of Hutter & Warner (1967). Its composition (in mm) was NaCl 450, KCl 8, CaCl2 20, MgC12 12, N-acetylglycine 2, and Tris maleate 2. The solution was brought to ph 8-0 by adding NaOH. In some experiments the osmolality was varied by the addition of sucrose or NaCl or by reducing the concentration of NaCl. To lower the concentration of C1, Na methanesulphonate was substituted for NaCl. The concentration of K was increased in one of three ways (Boyle & Conway, 1941): adding KCl to the control solution, isosmolal substitution of KCl for NaCl, or increasing the K concentration so as to keep the product [K]0. [Cl]0 constant. In selected experiments the ph was lowered to PVP was brought to the desired ph with NaOH, the osmolality being adjusted by appropriate changes in the concentration of NaCl. RESULTS Response of cell diameter and intracellular ion concentration to anisosmolal solutions For the purposes of this study we wished to measure the deviations from the isosmolal reference state brought about by small changes of the external K concentration above or below the physiological K concentration. An increase of the external K concentration above 18 mm would have resulted in contracture, thereby interfering with the diameter and membrane potential measurement; changes in cell diameter and volume occurring under physiological conditions are probably relatively small; and small deviations are more likely to be reversible. The measurement of such small deviations made it necessary to define rather closely the sensitivity of the assay systems and the stability or reversibility of the preparation and apparatus. For this purpose we made a preliminary study of the response to anisosmolal solutions which, in barnacle cells at ph 8-0, produce net movements of water but not of K and Cl. Response of cell diameter to anisosmolal solutions. Fig. 2 shows the response of the cell diameter to stepwise increases in the external osmolality. The osmolality was raised by adding sucrose in increments of 100 mm to a total of 100, 200 and 300 mm sucrose, respectively. The fibre diameter began to decrease promptly in response to each increase in osmolality. However, it required 20 min to approach a new stable value, the time constant (r) being 3-5 min. The direction of the change promptly reversed in response to re-perfusion with the sucrose-free control solution and approached the control value with a time constant of 3 min. The maximal decrease in diameter was 11 %, a much smaller maximal change than those of comparable studies on other muscles (Reuben et al. 1964; Blinks, 1965; Caputo, 1968; Gainer & Grundfest, 1968).

6 54 B. A. MOBLEY AND E. PAGE In other experiments the external osmolality was increased stepwise with NaCl. The degree and rate of shrinkage in response to hypertonic NaCl were like those in response to sucrose (r = 2-5 min). By contrast, cell swelling in response to a decrease in external osmolality was only partially reversible if the NaCl concentration of normal sea water was reduced by 33 mm or more. ph=79 0 _ S T=25' C E O U 0 X E * E * U 1000 _* * Time (min) Fig. 2. Response of cell diameter to stepwise increase in the external osmolality. The test solutions were made hypertonic by adding sucrose to isosmolal sea water to yield final sucrose concentrations of 100, 200 and 300 mm respectively. The osmolalities of the test solutions were 1056, 1198 and 1348 m-osmole/kg H20 respectively. The osmolality of the control sea water was 932 m-osmole/kg H20. Derivation of cell volume from diameter measurements. Although the most important conclusions from the diameter changes can be reached by considering only the time constants of these changes and the equilibrium values of the diameter approached with these time constants, it is also of interest to determine how the cell volume and the cell water content vary as a function of the osmolality. For this purpose it has been assumed that the cell volume is proportional to the square of the measured diameter. The diameter in the control sea water is denoted DC) and th estable diameter attained after a sufficiently long time in anisosmolal test solution is denoted DT. The osmolalities in control and test solutions are, respectively, Qc and QT. A plot of 100 D2jD 2 against loo nc/qt for reversible osmo-

7 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 55 lality changes in hypertonic solutions (Fig. 3) yields a least squares line, y = x. The graph represents the results of experiments on three fibres. The extrapolation of the line intercepts the ordinate at a value of 34-4 %. Fig. 3 suggests that of fibre volume in the control sea water fails to respond to an osmotic gradient. Comparable figures for other types of skeletal muscle are: 35 % for frog fibres (Reuben et al. 1963); 40 % for ~~~~~~~~~~ - X 60 Chi~~~~~~~~~.li,,,,,,/le 40-,,," 34.4-, QC x 100 Fig. 3. Determination of the osmotically inactive volume of barnacle muscle cells from measurements of the diameter of cells in control sea water and hypertonic test solutions. The different symbols represent different barnacles. A least-squares regression line for the points is y = x. The extrapolation of this line intercepts the ordinate at a value of The outer dashed lines are each three standard errors of the mean from the least-squares line (3 x 0-3 %). The standard deviation of the intercept is %. The correlation coefficient is crayfish fibres (Reuben et al. 1964); and 35 0/ for crab fibres (Hays et al. 1968). Gainer & Grundfest (1968) obtained a figure of only 10 % for lobster fibres. Assuming that the osmotically unresponsive volume is constant under all of the experimental conditions of this paper, the ratio (cell water in test solution)/(cell water in control solution) is given by (cell water)t/(cell water)c = (DI-0-344D')/(D-0344DD). (1) This relation will be used in subsequent sections to compute the cell water

8 56 B. A. MOBLEY AND E. PAGE content from the diameter measurements. The ratio (cell water)t/(cell water)c x 100 will be referred to as the calculated cell water (in units of per cent). In our own experiments and in those of others (Dydynska, & Wilkie, 1963; Hays et al. 1968) the use of the intercept (as in Fig. 3) to obtain the osmotically unresponsive fraction of fibre volume depends on the validity of the two assumptions that the volume is proportional to the square of the measured diameter and that the behaviour of cell volume at high osmolalities can be approximated by a linear extrapolation using data obtained from relatively small volume excursions around the isotonic value. It will be shown in the next section that, for our own data, the first assumption is TABLE 1. Cellular water content and intracellular K and Cl contents and concentrations in control sea water* Kg water/kg dry wt. Intracellular e A + Cell ion content concentration Water in (m-mole/kg dry wt.) (m-mole/kg cell water) sucrose Cell - Al space water K Cl K Cl * *02 +0* * Data are mean values for twenty fibres from four barnacles. a reasonable approximation. The extrapolation itself is a conventional one which has been widely used in the literature on cell volume in muscle and other tissues. The validity of the extrapolation has not, however, been independently established. We have therefore thought it appropriate to present the data on the calculated cell water content (which is based on this extrapolation) and that on cell diameter (which is independent of the extrapolation). Chemical determination of cell water and ion contents under control and anisosmolal conditions. Provided that cell volume is indeed proportional to the square of the measured diameter, it should be possible to confirm the intercept in Fig. 3 (based on measured diameters) by measuring cell water content gravimetrically and using the sucrose space to correct for extracellular solution. Tables 1 and 2 give the water contents and the intracellular ionic concentrations under control and anisosmolal conditions. The quantity R (Ve/dry wt.) + 1 (cell water/dry wt.) + (Ve/dry wt.) + 1' where Ve is the volume of solution in the sucrose space, dry wt. is the dry weight of the fibre corrected for the weight of salt in the sucrose space, and

9 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 57 V+l+l+l+l+l+l+l+l+l e~~~~+ O Cs ~~ Z ~~~ 00 t- C ~( 00 _ 1I-Ite v2 o C ~~~+l+l+l+l+l e X 0e e 0:e o( s +l+l+l+l ct *- r c :~~~~~~ Uoe Nt t M O e ~~~~+l+l +l +l +l +l +l +l +l C._ ; a X b o o z aq "i c CA U' xf If lf o mp e:> > to I CI ~~~~~~~~C C) CsCn_C> Ce C C c fi U e +l~~~+ +l +li +li +I +I +l +li +I> -1Z~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 ; G) ~~~~~~~~~~~~~~~~~~~o c l 4z 0 ; - _ 6e c6 6 fl ; X - ^ o o4 4 o E- to 16 ; o ad X > O C o o o o oc o U t s C) h X t < > > b 6 4 _o t- cc 0 t- 11 C - 0 o_ *C C C C C C m t _ o ~ ~~~~ 00tC Q 0".-( V O s O o. C C C. _ Fn+ _ I_ ,_ e. tt "et "I 1- vt m c3 e

10 58 B. A. MOBLEY AND E. PAGE 1 is the fraction dry wt./dry wt., was determined for each cell of Table 1. R should give the fraction of the total volume of a fibre in control sea water contributed by the sucrose space and dry solids. The mean value of R for the twenty cells is % (S.E.), a figure which is very close to that of 34-4 %, the osmotically inactive volume determined from the diameter measurements. The agreement suggests that the measured diameter can be used to approximate the cell water content according to eqn. (1). These results also suggest that the deviation from a cylindrical shape considered by Blinks (1965) do not seriously affect the conclusions about cell volume derived from the diameter measurements, at least over the range of departures from isotonicity examined by us. The changes in water content and intracellular ionic concentrations shown in Table 2 are in the expected directions (Adrian, 1956). They confirm that the observed changes in fibre diameter represent shifts in cell water content. Effects of varying the concentrations of K and Cl at ph 8-0 Hodgkin & Horowicz have shown that in frog muscle the response to an increase in external K concentration at constant Cl concentration is biphasic: a rapid initial depolarization is followed by a slow secondary response. The completion of the rapid initial phase of depolarization signifies the establishment of a diffusion potential across the membrane in accordance with the relative permeabilities of the membrane to K and Cl; the slow secondary phase reflects the net movements of KCl and water produced by the disturbance in the equilibrium distribution that prevailed before the increase in external K concentration. The following experiments show that in barnacle muscle at ph 8-0 the initial depolarization is unexpectedly slow, and that the secondary slow phase is absent. Fig. 4 shows the time course of the cell diameter and calculated cell water when the [K]o was raised from 8 to 18 mm by isosmolal substitution of KCl for NaCl. During 320 min in the test solution the cell water content increased by only 0-9 0/ S.D. (n = 3, control; n = 7, test) above the control value. This change, which is near the statistical limits of detection of the diameter measurement, is in the direction expected from the theory of Boyle & Conway (1941). If the absolute permeability of the membrane of barnacle muscle to KC1 at ph 8-0 is indeed as low as suggested by Fig. 4, measurements of the membrane potential under the same conditions ought to show that the slow secondary voltage transients described by Hodgkin & Horowicz in frog muscle are absent in barnacle muscle. The membrane potential measurements in Fig. 5 show that no slow membrane potential transient is evident on raising [K]0 from 8 to 18 mm by isosmolal substitution of KCI

11 K AND C1 ON VOLUME AND MEMBRANE POTENTIAL 59 for NaCl or on lowering it from 8 to 4 mm by isosmolal substitution of NaCl for KCl. An additional experiment, in which the fibre was kept in the high K solution for 55 min, showed no slow secondary membrane potential transient. In another experiment [K]o was reduced from 8 to 1 mm, then raised again to 8 mm; again no transient consistent with a net movement of KCl and water was noted. These experiments also illustrate two noteworthy features of the membrane potential response to altered [K]o at constant [Cl]0. (1) The time constant characterizing the approach to a stable new potential was larger KCI KCI 8 mm, 18 mm, NaCI NaCI s 450 mm 440 mm.1 8///8//8/ E -~~~~~~~~~100.0 C:IC! S10 -I ph=8*0 =- I T=23-24 C Q Q X X~~~X X X X X x X U U.~~ i xx xx S. 2 for Na0l than that in other single, cell preparations of muscle for comparable changes in [K]0 (Hodgkin & Horowicz, 1959; Zachar, Zacharova & Hencek, 1964). In the experiment of Fig. 5 a clear-cut depolarization was apparent within 10sec after changing [K]0 from 8 to 18mm; nevertheless, the approach to a new stable value of 52 mv was slow (r = 0i5 m). On re-perfusion with the control solution, repolarization was even slower (t = 1-5 min). Similarly, the depolarization on the increase of [K]o from 4 mm and the hyperpolarization on return to the low [K]o solution were both slow. (2) The initial response to an increase in [K]o was faster than that for a decrease.

12 60 B. A. MOBLEY AND E. PAGE Fig. 6 is a semilogarithmic plot of the membrane potential against [K]0. The potentials were measured in three fibres from three different barnacles. The values used in Fig. 6 were the stable potentials approached after a sufficiently long time following the change in [K]0. The barnacle membrane at ph 8-0 is an unusually good K electrode even at K concentrations much lower than those which the animal encounters under physiological conditions. KCI KCI KCI KCI KCI KCI 8mM, 18mM, 8mM, 4mM, 8mM, 4mM, NaCI NaCI NaCI NaCI NaCI NaCI 450 mm 440 mm 450 mm 454 mm 450 mm 454 mm ph= T= C so Time (min) Fig. 5. Time course of the membrane potential at ph 8-0 of a cell in test solutions of [K]o = 18 and 4 mm. [K]0 = 8 mm in control sea water. If the permeability to Cl (relative to that of K) is very low, at ph 8-0, it is to be expected that a lowering of [Cl]o at constant [K]o will fail to produce a significant depolarization. This expectation was confirmed: in three of four experiments a decrease in [Cl]o from 522 to 72 mm at constant [K]o produced no measurable depolarization even after long times; a fourth experiment showed a depolarization of only 4 mv in 50 min. Effects of varying the concentrations of K and Cl at ph 4-5 Hagiwara, Gruener, Hayashi, Sakata & Grinnell (1968) demonstrated that, relative to its value at ph 7.7, the total membrane resistance decreases sharply when the ph of the solution bathing a barnacle fibre is lowered from 5.0 to 4 0. They were able to show that the fall in total resist-

13 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 61 ance results from a selective increase in the Cl conductance. They found that at ph 4 the ratio (Cl conductance)/(k conductance) was 6-9, compared to a value of at ph 7-7. We have taken advantage of this observation by Hagiwara et al. to examine the transients in cell volume and membrane potential produced by net movements of K and Cl under conditions in which the absolute x > 80 -t A I, [K] 0 (mm) Semilogarithmic plot of the steady-state membrane potentials at Fig. 6. ph 8*0 against the external K concentration. The different symbols represent different fibres from different barnacles. The dashed line is the K equilibrium potential. RT7 VK = IIn ([K]0/[K], with [K1] (Table 1), T = 250C. = 197 m-mole/kg H20 permeability to KCl is greater than at the ph ofnormal sea water. Between ph 4*4 and 4*6 the Cl permeability is large enough so that significant net movements of KCI and water become apparent. In Fig. 7 the experimental conditions were similar to those of Fig. 4, except that the experiment was done at ph 4*5 instead of at ph 8-0. It is apparent from Fig. 7 that raising [K]o from 8 to 18 mm by isosmolal substitution of KCl for NaCl brings about a significant increase of 300/ + 0*2 (S.D.) (n = 3, control; n = 10, test) in cell water content in about 200 min (T = 115 min). Although the total increase in volume was small, the swelling was not reversible: upon re-perfusion with control solution, the calculated cell water content decreased from 103-0% of control to %,

14 KCI KCI KCI 8mM, 18mM, 8mM, NaCI NaCI NaCI g 4SO mm 440 mm 450 mm Of ~~1 62 B. A. MOBLEY AND E. PAGE a decrease of 0.7 /% (S.D.) (n = 10, test; n = 13, control). Two additional experiments gave similar increases in cell volume, although reversibility was not examined. In a further experiment [K]0 was reduced from 8 to 1 mm by isosmolal substitution of NaCl for KC1. Under these conditions one might expect a net outward movement of KCI and therefore a reduction of cell water content. No such reduction was observed after more than 200 min in the 1 mm-k solution ] l ~~XX XXMXpH=45S I nx--iixzxx~ K x T= C XI Xx 1xxx xxxi x ~~~X o I o o 0~~0~ ~ *0000o U ,,, L -'.5 MO"o" ooo I I I Time (min) Fig. 7. Time course of the cell diameter and calculated cell water at ph 4-5 when [K]o was raised from 8 to for NaC1. 18 mm by isosmolal substitution of KCl Fig. 8 shows the change in membrane potential under conditions identical Raising [K]o from 8 to 18 mm produced a measurable to those of Fig. 7. depolarization within 10 sec; the change from 72 to 59 mv required 33 min after introduction of the test solution. During the next 90 min the membrane depolarized by an additional 1.5 mv. The secondary phase of slow depolarization corresponds in duration to the slow increase in cell water content shown in Fig. 7. Upon changing the bath back to the control solution the fibre partially repolarized; the membrane potential returned to a value of 66 mv (r = 2-5 min). The reversal of the depolarization, like that of the change in cell volume, was incomplete. Figs. 7 and 8 and related experiments not illustrated were interpreted as showing that at ph 4-5 the cells take up KCI and water when [K]o is raised

15 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 63 isosmotically at constant [Cl]0, but that KCl fails to move out of the cells (or moves out extremely slowly) in response to a lowering of [K]0. These results suggest that barnacle muscle manifests the phenomenon of anomalous rectification which has been described in frog skeletal muscle on the basis of electrophysiological measurements (Katz, 1949; Hodgkin & Horowicz, 1959; Adrian & Freygang, 1962) and of chemical measurements (Adrian, 1960). Figs. 9 and 10 are separate experiments showing the respective time courses of the cell water content and membrane potential in response to the KCI KCI KCI 8 mm, 18 mm, 8 mm, NaCI NaCI NaCI 450 mm 440 mm 450 mm E ph =45-46 T=23' C 60- Fig I I Time (min) Time course of the membrane potential at ph 4-5 of a cell in a test = 8 mm in control sea water. solution of [K]0 = 18 mm. [K]0 isosmolal reduction of [Cl]o at constant [K]o. [Cl]o was lowered from 522 to 72 mm by isosmolal substitution of Na methanesulphonate for NaCl. The cell water content decreased progressively by 3-4 % ± 0-2 (S.D.) (n = 4, control; n = 8, test) in about 350 min (T = 155 min). The membrane potential fell measurably within 10 sec. Fifteen minutes were required for a depolarization from 76 to 58 mv (T 1 min), and then the membrane slowly repolarized to 63'5 mv during the next 45 min (T = 17 5 min). Upon re-perfusion with control solution, there was a further rapid repolarization to 73 mv within 10 min (T = 0.5 min). In this and a second similar experiment no hyperpolarization was observed on re-perfusion with the control solution. PHY 2I5

16 EI. la 1200 B. A. MOBLEY AND E. PAGE [CI] 522 mm t xx [CI] 72mM xx xxi XXXX A -1 7v X ---. I.-I L. X 1X0Hb 4U~* * % X 98.8 ph=4*6 T= C x * 00 w 95S0- - u Soo 6CDO Time (min) Fig. 9. Time course of the cell diameter and calculated cell water at ph 4-6 when [Cl]0 was lowered from 522 to 72 mm by isosmolal substitution of Na methanesulphonate for NaCl. [CI] 522 mm 80 [CI] 72 mm... [CI] 522 mm i ~~~~~~~~~~~I 5~ ph-=446 T=22-23 C Time (min) Fig. 10. Time course of the membrane potential at ph 4-6 of a cell in a test solution of [Cl]. = 72 mmx. [Cl]o = 522 mm in control sea water.

17 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 65 In accordance with the interpretation of Hodgkin & Horowicz (1959), we attribute the loss of cell water and the slow secondary repolarization in low Cl solution to a loss of KCl from the cells in response to the displacement of the Cl equilibrium potential. The fibre of Fig. 10 was not in the test solution long enough to lose more than a small amount of KCI and water. For this reason, and also because of anomalous rectification, a hyperpolarization was not expected upon re-perfusion with the control solution. If [K]0 and [Cl]0 are changed in such a way as to keep the product [K]o. [Cl]o constant the membrane potential should change without associated net movements of ions and water into or out of the cell. We have therefore examined the respective responses of cell water content and membrane potential in experiments in which [K]0 was increased from 8 to 18 mm at constant [K]o. [Cl]o. No net gain or loss of water attributable to a KCl influx or efflux was observed. The membrane potential fell measurably within 10 sec from its control value of 73 mv: it approached a value of 57 mv (T = 2 min) and then remained constant. This change was reversible after re-perfusion with control solution (r = 2 25 min). An experiment in which the fibre was left in an identical test solution for a much longer time showed that the membrane potential remained stable at its maximally depolarized value for 52 min. This experiment is noteworthy for the relative slowness of the depolarization. If the depolarization depended only on the change of K and Cl concentrations at the external (cylindrical) surface of the cell, the change in membrane potential should have been completed in the few seconds required for mixing within the chamber. Experiments to determine the origin of the delayed depolarization in response to raising [K]0 On the basis of the experiments so far described, we had observed a consistently large time constant for the response of the membrane potential to changes in K or Cl ions. We considered three possible explanations for this phenomenon: (1) an intermediate permeability barrier interposed between the K-responsive cell membrane and the bathing solution - for example, a barrier at the level of the basement membrane; (2) a delay due to the slowness of diffusion of KCl within the system of invaginations and T-tubules; and (3) a non-uniform response of cell water to an osmotic gradient imposed across the cell membrane because intracellular mixing (by diffusion) is not instantaneous relative to the rate of net movement of water across the cell membrane. Although it would seem to be mainly responsible for the long times (T = 2-5 min) needed for the fibre to approach a steady state after exposure to an anisosmolal solution, the 3-2

18 66 B. A. MOBLEY AND E. PAGE third explanation cannot account for the delayed membrane potential response observed when [K]0 is changed at ph 8-0 or when [K]o and [Cl]0 are changed at constant [K]0. [Cl]o: under these conditions, there are little or no net movements of water or ions across the cell membrane. We therefore had to distinguish between an effect due to an intermediate permeability barrier and an effect due to delayed equilibration within the system of invaginations and T-tubules. The experiments designed to make this distinction were based on the fact that the rate of diffusion in free solution is, to a first approximation, inversely proportional to the viscosity. If it were possible to increase the viscosity within the system of invaginations and T-tubules and at the same time to bathe the external (cylindrical) cell surface with a depolarizing solution of normal viscosity, it should be possible to slow diffusion of K in the invaginations and T-tubules and thus to determine the relative contributions of the external surface and the system of invaginations and T-tubules to the magnitude and time course of a K-induced depolarization. In Fig. 11 the membrane potential was 73 mv in control sea water; perfusion with a sea water of the same K concentration containing 10 % PVP (average molecular weight 40,000) caused an immediate depolarization of about 2 mv. The fibre was kept in PVP sea water for 2-5 hr, then perfused with sea water containing 18 mm-k. The high K solution produced an early depolarization (r = 0 4 min), but this depolarization was only about 6 mv. The initial depolarization was followed by a secondary repolarization of about 3 mv which was maximal about 5 min after the start of the perfusion with 18 mm-k. The membrane then depolarized to 59 mv over a 32 min period (r - 13 min). Upon re-perfusion with normal sea water, repolarization occurred with a nearly normal time constant of 1-3 min. A near normal response to increased [K]o was observed after perfusing the fibre with control sea water for 1 hr. A second experiment gave essentially identical results. We attribute the initial depolarization in 18 mm-k (after pre-equilibration with PVP) mainly to the depolarization of the cell surface exclusive of the system of invaginations and T-tubules; the time constant reflects the time required to mix the depolarizing solution with the highviscosity PVP sea water within the chamber. The slow secondary depolarization would then reflect the depolarization of the membrane lining the sarcolemmal invaginations and T-tubules within which the presence of PVP slows the diffusion of K. Repolarization is much faster than depolarization because PVP has diffused out of these channels during the exposure to the PVP-free depolarizing solution. The fact that after equilibration with PVP there is an initial rapid depolarization shows that PVP itself

19 K AND Cl ON VOLUME AND MEMBRANE POTENTIAL 67 does not act as a significant permeability barrier on the membrane covering the external surface. The small, transient repolarization which follows the initial depolarization could be prevented by including PVP in both the control sea-water and the depolarizing solution. Under these conditions, we observed only a small and rapid initial depolarization, followed by a secondary depolarization with an extraordinarily long time constant characteristic of cells equilibrated with PVP. We therefore attributed the small transient repolarization of Fig. 11 to a specific depolarizing effect of PVP on the membrane which is unmasked if the PVP is washed away by perfusion with a PVP-free solution. 90 E r PVP=10% [K]o=8 mm EKL= K]=mm [K]0=8 mm [K].=18 mm Time (min) Fig. 11. Effect of prolonged PVP perfusion on the response of the membrane potential to increased [K]O. We tested our interpretation that PVP slows diffusion of K in the sarcolemmal invaginations and T-tubules by repeating the experiment with a PVP polymer (molecular weight 160,000, 4 % concentration) so large that it cannot diffuse into these channels. Under these conditions only one time constant for depolarization (T = 08 min) was observed when fibres which had been pre-equilibrated for 2-5 hr in PVP-sea water were perfused with the PVP-free depolarizing solution. The prolonged depolarization which occurs in the presence of PVP of low molecular weight is thus absent in the presence of high-molecular-weight PVP; there is only a slight prolongation of the normal time constant, presumably because the high viscosity of the high-molecular-weight PVP slows mixing within the muscle chamber. DISCUSSION The experiments described in this paper show that the diameter of barnacle muscle cells indicates the time course of net ionic movements at least as sensitively as the membrane potential. The application of a sensitive technique to an unusually large muscle cell has made it possible to

20 68 B. A. MOBLEY AND E. PAGE follow net ion movements in the physiological range of external K concentrations. This range is normally inaccessible to experiments in other preparations, a circumstance which has often forced previous workers to employ K concentrations high enough to result in contractures. The diameter measurement can also be used to confirm that anomalous rectification occurs in barnacle muscle. Perhaps the most striking finding of these experiments was that the prolongation of depolarization and repolarization due to K and Cl seems to reflect the time course of diffusion within the system of sarcolemmal invaginations and T-tubules. There are two experimental observations which support this interpretation. (a) The finding that depolarization by K ion can be markedly slowed by introducing PVP into the sarcolemmal invaginations and T-tubules and (b) the asymmetry between the rates at which the membrane potential changes in response to increasing and decreasing the external K concentration. A similar asymmetry has previously been recorded in frog muscle by Hodgkin & Horowicz (1960) and attributed to the presence of the T-system. The distinctness of the effects of the sarcolemmal invaginations and T- tubules in barnacle muscle results from the large fibre diameter, which causes the ratio of the outer (cylindrical) surface to the total cell volume to be small; the low absolute permeability to K and Cl at ph 80; and the high ratio of the area of sarcolemmal infoldings and T-tubules to outer (cylindrical) membrane, which is estimated at about 20 by Selverston (1967). Permeability of K and Cl From our membrane potential measurements we have calculated the ratio (Cl conductance)/(k conductance) by the method of Hodgkin & Horowicz (1959). This calculation yields a maximal value of 1/12 at ph 8.0, a figure somewhat smaller than that of 1/6-1/7 obtained by Hagiwara et al. (1968). At ph 4-5 the ratio rises to a minimum of 1/2. At ph 8-0 the Cl conductance is rate-limiting for net movements of KCI across the membrane. The fact that such net movements become fast enough to be detected at ph 4-5 suggests that the increase in the ratio results from an increase in the Cl conductance as proposed by Hagiwara et al. We are indebted to Dr C. S. Spyropoulos for helpful discussions. We would also like to thank Mr George Gibson for constructing the muscle chambers. Portions of this work were presented by B. A. Mobley to the faculty of the Division of the Biological Sciences of the University of Chicago in partial fulfilment of the requirements for the degree Doctor of Philosophy in Physiology. This work was supported by USPHS Grant No. HE and by a grant from the American Heart Association. Dr Page is the recipient of a USPHS Career Development Award. Dr Mobley was supported by Physiology training grant USPHS GM

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