J. Phygiol. (1976), 256, pp. 663-689 With 14 text-figure8 Printed in Great Britain

663

INFLUENCE OF CHANGES IN EXTERNAL POTASSIUM AND CHLORIDE IONS ON MEMBRANE POTENTIAL AND INTRACELLULAR POTASSIUM ION ACTIVITY IN RABBIT VENTRICULAR MUSCLE

BY HARRY A. FOZZARD AND CHIN OK LEE* From the Departments of Medicine and The Pharmacological and Physiological Sciences University of Chicago, Chicago, Illinois, U.S.A.

(Received 26 August 1975) SUMMARY

1. The membrane responses of rabbit papillary muscles to rapid changes in [K]0 and [Cl]0 were measured with open-tipped micropipettes and with closed micropipettes made from K-selective glass. 2. The muscle cells behaved primarily as a K electrode, and responses to changes in [K]0 with constant [Cl]0 or with constant [K]0 x [Cl]0 were substantially the same. 3. When [Cl]0 was changed at a constant [K]0 the membrane potentials changed rapidly and symmetrically by a small value and remained constant for 30 min. 4. Measurement of potential with K+-selective micro-electrodes in these experiments showed no change in intracellular K activity. In addition to permitting calculation of K permeability, these measurements reassured us that the K+-selective electrodes were well insulated and not influenced by electrical shunts at the impalement site. 5. Although the membrane response to changes in [Cl]o was small, it was possible to calculate that the permeability ratio (Pc1/PK), was 04I1. The Cl and K conductances were about 0-015 mmho/cm2 and 0 09 mmho/ cm2 respectively, resulting in a conductance ratio (gcl/gK) of about 0417. 6. The time course of depolarization by increase in [K]o was rapid (half-time 5 sec), but repolarization on return to lower [K]0 was much slower (half-time 50 sec). The depolarization time course was easily fitted by the potential change calculated by assuming the need for K diffusion into the extracellular spaces and taking account of the logarithmic relation between membrane potential and [K]0. These calculations did not fit the time course * Send request for reprints to Dr C. 0. Lee, Department of Medicine, 950 East 59th Street, Chicago, Illinois, 60637, U.S.A.

664 H. A. FOZZARD AND C. 0. LEE of repolarization, which was slowed in the fashion expected from an inwardrectifying membrane. 7. The influence of [K]i on membrane potential was investigated by changes in tonicity of the external solution. Hypotonic solution produced a change in intracellular K activity close to that produced by ideal water movement. However, in hypertonic solution, intracellular K activity did not rise as much as predicted, suggesting a change in intracellular activity coefficient. INTRODUCTION

Hodgkin & Horowicz (1959) have shown that the membrane potential of frog skeletal muscle fibres is affected by both K and C1 ions, and the membrane can be made to behave either as a K electrode or as a Cl electrode under appropriate conditions. The relative roles of K and Cl permeabilities in cardiac muscle cells have been studied less than in skeletal muscle cells. The goal of this work was designed to investigate the effects of change in external K and Cl concentrations on the membrane potentials and intracellular K ion activities of rabbit papillary muscle cells and to determine the relative permeabilities and conductances of the two ions. In addition, internal K concentration was changed by altering the osmolarity of the external solution. Carmeliet ( 1961) has reported that Cl ion has little influence on the resting membrane potential at normal and higher K concentration. Hutter & Noble (1961) found that cardiac muscle cells are more permeable to K than to C1. However, in order to obtain detailed information about these permeabilities the membrane potentials should be continuously measured during sudden changes of the external solutions. It is also desirable to monitor the intracellular K ion activities during the changes. In these experiments the external solutions around the papillary muscle could be changed within a second with an apparatus modified from that used by Gibbons & Fozzard (1971). The intracellular K ion activities were measured with K+-selective glass micro-electrodes similar to those used in our earlier study on heart muscle cells (Lee & Fozzard, 1975a). In the application of K+-selective glass micro-electrodes to the relatively small cells, it was found that the shunts produced by incomplete sealing of the cell membrane around the micro-electrodes are not different from conventional open-tip micro-electrodes. This was indicated by the continuous measurement of membrane potentials with the micro-electrodes during the changes of external K concentration. Similar continuous measurements with recessed-tip Na+-sensitive micro-electrodes have been made in snail neurones (Thomas, 1972). The interpretation on the membrane potential changes after an altera-

665 K AND Cl EFFECTS ON MEMBRANE VOLTAGE tion of external K or C concentration is complicated by diffusion in the extracellular space of the papillary muscles with diameter of about 1 mm. However, it is clear that K permeability is much greater than Cl permeability and the cell membrane behaves primarily as a potassium electrode. These results in the cardiac muscle are different from those in frog skeletal muscle. Some observations have been reported (Lee & Fozzard, 1975b). METHODS Papillary muscles from rabbit right ventricles were used throughout this experiment. The muscles were about 1 mm in diameter and 5 mm in length. The apparatus, modified from that used by Gibbons & Fozzard (1971), is illustrated in Fig. 1. The papillary muscles were mounted in a narrow channel in the muscle chamber, into which solutions of different composition could be introduced via a valve. Dead space Micro-electrode

Groove for GIReference electrode overflow of GlassM solutions

Inlets for solutions

-

Rubber tubing

Outlet

Fig. 1. Diagram of muscle chamber for solution change. was minimized by positioning the valve close to the muscle. The tendon at tapering end of the muscle was fixed by a pin to paraffin, which filled a hole at the bottom of the channel. The other end of the muscles also was fixed by another pin to paraffin. As shown in the Figure, a saturated KCI salt bridge with 3 % agar was used as a reference electrode. The valve was connected to two rubber tubings for inlet of solutions with different compositions. The rubber tubings were connected to reservoirs mounted over the muscle chamber, where the solutions were vigorously oxygenated for about an hour. One reservoir contained normal Tyrode solution and the other contained test solution. After the muscle was equilibrated in normal Tyrode solution for about 30 min, cell membrane potentials were measured. When the membrane potential was stable for 1 min, the valve was turned to produce a sudden change of solution. Solution flow rate during exposure to a test solution was usually 1.5-1.8 ml./sec, corresponding to a linear solution velocity of 1-7-2-0 cshisec. in the channel of the muscle chamber. A linear solution velocity in this range should

H. A. FOZZARD AND C. 0. LEE

666

change the solution in the region occupied by a 5 mm long muscle in about 0 3 sec. A test of the rate of solution change was made in the fashion described by Gibbons & Fozzard (1971), and it was complete in about 0 5 sec. In order to facilitate removal of the waste solution during this fast flow, a groove with a width of 1 cm was made on the wall between the left compartment and the middle compartment, and the level of bottom of the groove was the same as that of the solution as shown by the interrupted line. In addition, four siphon tubes were mounted to accelerate overflow of the solution. Membrane potentials were recorded between a micro-electrode filled with 3 M-KCl and the agar reference electrode. Conventional micro-electrodes had tip potentials less than 10 mV and resistances of 20-50 Mn. The micro-electrodes were connected through an agar bridge to a calomel half-cell and the agar reference electrode was connected to another calomel half-cell. The calomel half-cells were connected to the input of a voltmeter (Keithly Instruments, Inc., Cleveland, Ohio; Model 610C). The potential difference between the two calomel half-cells was less than 0 5 mV throughout this experiment. Therefore, we could measure net tip potentials or junction potentials and their changes without breaking the micro-electrode's tip during the measurements of cell membrane potentials. The liquid junction potentials between the different test solutions were corrected to obtain accurate membrane potentials. This procedure will be seen in the results of each experiment. The construction of K+-selective glass micro-electrodes was similar to that described previously (Lee & Armstrong, 1974). Some requirements for use in small cells, such as cardiac muscle cells, appeared previously (Lee & Fozzard, 1975a). The resistance of the micro-electrodes used in this study was less than 500 Mn. This low resistance could be obtained by soaking the micro-electrodes in 3 M-KC1 for more than one week (Lee & Fozzard, 1974). TABLE 1. Composition of solutions

K+ A B C D E

F G H

(mM) 5-4 30 10 5-4

5.4 30 5-4 5-4

Na+ (mM) 153 128 148 153 149-4 128 113 153

Cl(mM) 148-2 148-2 148-2 73 73 26-6 108 148-2

Pr(mM)

75 75 124

Ca2+ (mM) 1-8 1-8 1-8 1-8 3-6 1-8 1-8 1-8

Sucrose (mM) 11.1 11.1 11.1 11'1

Relative tonicity 1.0

11 1 11.1

11-1 179

In addition to the above, the solutions each contained: Mg2+, 1-1 mm: 13-5 mM: H2PO4-, 2-4 mM.

1-0 1-0 1-0 1-0 1.0 0-76 1-5

HCO3-,

The external solutions and their composition are listed in Table 1. The Tyrode solution, A, is identical to that used by Gibbons & Fozzard (1971). This solution was used during dissection of papillary muscles and equilibration of the muscles before measurements of membrane potentials. The solutions, B and C, were used in the experiments of effect on membrane potential of high external K concentration with constant external Cl concentration. These solutions were made by substituting equimolar KCl for NaCl. The solution D was used to reduce Cl concentration at constant external K concentration. This solution was made by substituting equimolar Na propionate for NaCl. Solution E was used for similar purpose to the

K AND Cl EFFECTS ON MEMBRANE VOLTAGE

667

solution D except external Ca concentration was 3-6 mm instead of 1-8 mm. The solution F had a constant K-Cl product of 800 (mM)2. The concentrations of K and Cl in the solutions, A and F, were 5-4-148-2 mmz and 30-26-6 mm respectively. The solution F was made by using Na propionate and K propionate. The solution G was used for the hypotonic solution. This solution was made by reducing NaCl to 40 mM, where relative tonicity is equivalent to 0 76. The solution H was the hypertonic solution, with relative tonicity of 1-5. This solution was made by adding dextrose of 168 mm. The solutions listed in Table 1 have constant ionic strength except the solution G. The solutions had pH of about 7-2 when they were saturated with a gas mixture of 95% 02 and 5O% CO2. A

30 mM

30 mM

K55 148 mm C1

-3-

mV -34-2,lV-

-50 50L)

0

K.5-4

1 2 30 mM

__

__

-rt 358 mV -34 9

2o 1

2

_

__

_

__

_

__

_

148 mmCIm -100 -75-8 mV

-76-3 mV

0

LL~

6-1

CI

38

-so

-100

3 10 11 12 13 14 15 16 17 Time (min) 30 mM 5-4 mM K

148 mm

0

mV

5-9 2-6

7-4

mV

-74-6 mV

-75-7 mV

-100

MV -100 -50

C

148 mM

mV

B

K

m

mV

-

4-3

0-2 0.5 3

4

O

60 61 62 63 64 65 66 67 68 Time (min)

Fig. 2. Effect on membrane potential of changing [K]o from 5-4 to 30 mm and from 30 to 5-4 mm with constant [Cl]0. In A, a papillary muscle with diameter of about 1 mm remained in 30 mM-K for about 5 min between two continuous measurements. In B, a papillary muscle with diameter of about 1-2 mm remained in 30 mM-K for about 1 hr between two continuous measurements. The small numbers before and after cell entry are the tip

potentials. RESULTS

Variation of external K at constant external Cl Fig. 2 illustrates the effects of a change in [K]0 on the membrane potential of rabbit ventricular muscle cells at constant [Cl]o. The normal Tyrode solution (solution A of Table 1) contained 5'4 mm-K and 148*2 mMCl and the test solution (solution B of Table 1) contained 30 mM-K and 148*2 mM-Cl. When the test solution was applied (Fig. 2A), the resting membrane potential of - 75-7 mV first fell rapidly and then reached a

668 H. A. FOZZARD AND C. 0. LEE stable potential of - 34-2 mV close to its equilibrium potential of -34 mV. The fall from the resting membrane potential to the equilibrium potential took about 1.5 min for a papillary muscle with a diameter of 1.0 mm. The values (7.4 and 5.9) shown at the beginning and the end of the potential recordings indicate the absolute value of the micro-electrode tip potential before impalement and after withdrawal. The membrane potentials of - 75-7 and - 34-2 mV are already corrected and so do not include the tip potential. Since tip potentials usually change when the external solution is changed, they must be measured and corrected. This procedure will be followed throughout this work. The right part of Fig. 2A shows the time course of repolarization on restoring the original solution after 5 min in the 30 mM-K solution. The membrane potential changed from the equilibrium potential of - 33.9 mV to a resting potential of - 74-6 mV. This repolarization is slower than the depolarization, taking about 4 mi to restore the resting potential. Fig. 2B shows similar experiments to A except a papillary muscle with diameter of 1x2 mm remained in 30 mM-K solution for about 1 hr. When 30 mM-K solution was applied, the potential rapidly decreased from the resting potential of - 76-3 to - 35-8 mV close to the equilibrium potential. It took about 2 min to reach the stable equilibrium potential. On restoring 5-4 mM-K solution after leaving the fibre in the test solution for about 1 hr, the potential increased slowly from - 34.9 to - 75 8 mV and it took about 5 min to recover the stable resting potential. The differences in time course of full depolarization and repolarization for the two fibres are small and might be expected from the diameters of the muscles. The 41 mV of depolarization and repolarization is close to the difference of about 42 mV between the resting membrane potentials in 5-4 and 30 mm external K concentrations (Lee & Fozzard, 1975a). These results obtained from cardiac muscles are different from those observed in frog skeletal muscles. First the time of full depolarization in the cardiac muscles was about 2 min whereas the time in the skeletal muscles was more than 40 min even though single fibres of the skeletal muscles were used. Secondly, the times of repolarization in cardiac muscle are similar regardless of the applied time of high K solution, whereas the times for repolarization in the skeletal muscles are markedly different. However, asymmetry between depolarization and repolarization was observed in both muscles (Hodgkin & Horowicz, 1960). The results described above suggested that the intracellular K ionic activity of the cardiac muscles is not altered by changes in the [K]o at constant [Cl]o. To test this suggestion, the intracellular K ionic activities during the changes in [K]o were measured with K+-selective glass microelectrodes. For the measurement of only the intracellular K ionic activity,

K AND Cl EFFECTS ON MEMBRANE VOLTAGE 669 it is necessary to characterize the K+-selective glass micro-electrodes for their selectivity and response time because of the following reasons: (1) the K+-selective micro-electrodes are usually sensitive to Na ions to some degree, (2) the micro-electrodes have high resistance and (3) cell membrane potentials must be measured with conventional micro-electrodes under the same conditions. Fig. 3 illustrates the selectivity and response time of the K+-selective micro-electrode. Fig. 3A shows calibration of the 30 mm

1 mM

-250 1 mm

-229-6 mV A 10mm -172-9mV

~200E-18O~mV 10 mm -150 -12431 mV > E 100mM 100 -70-2mV

K 148m

1

_144-2mV

100mM mm mV

I

-20

B

-1031

- 1 188

-150 mV

-76

E~m -5m

-100e

>

-50:0 1mmi KCI soln.

0 NaCI soin.

1

2 Time

3

4

5

(mi)

Fig. 3. A, calibration of a K-selective micro-electrode in KCl and NaCi solutions. The potential value (mV) for each solution was read from a Digitec voltmeter. B, change in transmembrane potentials simultaneously measured with K+iselective micro-electrode (a) and conventional micro-electrode (b) during sudden change in [K]0 at constant [Cl]0.

micro-electrode in KCl solutions of 1, 10 and 100 mm and NaCl solutions of 1, 10 and 100 mm. The behaviour ofK+aselective the micro-electrodes can be described according to the following equation: Ey, = EQ+ Slog (ay,+ kK.N.a. )N(1) where EK is micro-electrode potential; E0 is a constant of the microelectrode; S is an empirical slope obtained from calibration (AEK/A log aK); aK and aNa, are activities of K and Na ions; kKNa is selectivity coefficient of micro-electrode. E0, S and kKNa of eqn. (1) can be determined from the calibration data obtained in Fig. 3 A (Lee & Fozzard, 1974), and were - 5-7 mV, 58-1 mV and 0 154 respectively for this electrode. Using these values, when the micro-electrode potentials are plotted against the corresponding log aK and log aNa, one can obtain two parallel straight lines. These results indicate that the selectivity coefficient is constant in the range of the test concentrations of KCl and NaCl, and the micro-electrode is about seven times more sensitive to K ion than Na ion. The K+-selective micro-electrodes can be applied for the measurement of intracellular K ionic activity only when the term of kKNaaNa in equation 1 is small relative to the term aK in the equation. The micro-electrode used in Fig. 3, which had been stored in 3 M-KCl for about two weeks, had kKNa of

670 H. A. FOZZARD AND C. 0. LEE 0*154 and tip resistance of about 200 MQ. In this study only microelectrodes with kK, Na less than 0-2 were used to measure intracellular K ionic activity. In the cardiac muscle cells the intracellular Na ionic activity of 5*7 mm is smaller than the intracellular K ionic activity of 82'6 mm (Lee & Fozzard, 1975a). Thus kKNa.aNa is small relative to a] in eqn. (1) (about 1 %) and so one can use the following equation: EE = E0+Slog aK (2) When transmembrane potential is measured with a K+-selective microelectrode to obtain intracellular K activity, membrane potential measured with conventional micro-electrode (VM) should be subtracted from the transmembrane potential (EK). Thus eqn. (2) gives the following equation:

EK-VM

=

EO+S log aK.

(3)

When the intracellular K activities are measured with K+-selective microelectrodes during changes in membrane potential, the response time of the K+-selective micro-electrodes should be satisfactorily close to that of conventional micro-electrodes for measuring VM. As seen in the calibration of the micro-electrode in Fig. 3A, the response time in each solution is so fast that any delay in response time cannot be seen. Fig. 3B shows the time courses of membrane potential changes measured with the K+selective micro-electrode and a conventional micro-electrode during change in [K]o from 5*4 to 30 mm. In this experiment a papillary muscle with diameter of 10 mm was used. The tracing a represents the membrane potential change measured with the K+-selective micro-electrode calibrated in Fig. 3A. When the micro-electrode impaled a cell, a stable potential of - 144*2 mV was registered at [K]o of 5-4 mm. On applying 30 mM-K solution, the potential fell rapidly and then reached a stable potential of - 103-1 mV after about 1-5 min. The tracing labelled b represents the membrane potential change measured with a conventional micro-electrode in the same muscle. A resting potential of - 76-3 mV was registered and on applying 30 mM-K solution the potential fell in similar fashion with the tracing a. The time courses of the two potential changes are virtually parallel. In this time scale the response times of the two micro-electrodes do not differ, so that the intracellular K ionic activity can be satisfactorily measured during sudden changes of external solution. Also the results indicate that the intracellular K ionic activity does not change during depolarization produced by raising [K]0. Using the results obtained in Fig. 3, the intracellular K ionic activity calculated by eqn. (3) was 84-3 mM which is close to an average value of about 83 mm obtained from our earlier study (Lee, & Fozzard, 1975a). The effects of reducing [K]0 on the membrane potentials measured with K+-selective micro-electrodes are shown in Fig. 4. When 30 mM-K

K AND Cl EFFECTS ON MEMBRANE VOLTAGE 671 solution was applied during the third impalement, the membrane potential of - 145-6 mV fell rapidly and then reachedastable potential of - 104-2 mV after about 1-5 min. The time course of the depolarization and its magnitude of 41-4 mV are close to those in the depolarization of Fig. 2A which was measured with a conventional micro-electrode in the same muscle and 30 mM

KK

30 mm

30 mM

148 mm

1

-145 6 mV mV -143-4 -150 ' ' 'a il.V -992 mV _ E100 -50

-138 9 mV -98*1

mV

B 8

70 71 72 73 74 75 76 77 78 7980 Time (min) 30 mm K

l

148 mm

9mV -102-8-1019 _

-501-P

-100 -

o

0

-50

VA~~~~~~~~~~~~~~~~~~~

K Cl

-15

15S -100>

A 0 012 3 4 5 6 Time (min)

E

K

148 mm

1

Cl

-1418 mV

B'

-5

J50o5

-100>

2 3 4 5 6 7 8 9 10 11 Time (min)

Fig. 4. Effects of sudden increase and reduction in [K]0 on transmembrane potentials measured with a K+-selective micro-electrode. In A and B, a papillary muscle with diameter of about 1 mm was used. In B', a papillary muscle with diameter of about 1-4 mm was used. Hi and T indicate impalement and withdrawal of the micro-electrode.

condition. On restoration of 5-4 mM-K solution after the muscle remained in 30 mM-K solution for about 1 hr (panel B), the membrane potential increased from - 99-2 to - 138-9 mV. The full repolarization took about 4 min. The time course of this repolarization is virtually identical to that of the repolarization in Fig. 2A which was measured with conventional micro-electrodes. These results indicate that the intracellular K ionic activity did not change during repolarization produced by reducing [K]0 even after a long time in high K solution. On reapplying 30 mM-K solution as shown in Fig. 4B, the potential fell from - 138-9 to - 98-1 mV. This depolarization also is similar to those seen in Fig. 4A and Fig. 2A. However, the time course of the potential change in muscles of different diameter was different. An example of the variation was shown in Fig. 2A and B. in which the potential changes were measured with conventional micro-electrodes. This variation was also observed in the potential changes measured with K+-selective micro-electrodes, as shown in Fig. 4B'. In

H. A. FOZZARD AND C. 0. LEE 672 the second impalement, a membrane potential of - 101 9 mV was measured with a K+-selective micro-electrode in the muscle with diameter of 1P4 mm, which had remained in 30 mM-K solution for about 20 min. On restoring 5*4 mM-K solution, the potential increased at relatively slow rate and it took about 6'5 min for full rep. larization. This variation was not due to the time of exposure to the high K solution, producing ionic redistribution across the cell membrane since the full repolarization was complete in shorter period (about 4 min in Fig. 4B) in the muscle that remained in the high K solution for about 1 hr. This variation probably resulted from the effect of muscle diameter on K diffusion in the extracellular space. 10 mM 2 mem

K

-200

-1514 mV

-200 50l-1

~~~~~~~~~~~~-

m

-150O7mV1239V

-150 E

K

148 mm

-150

~~~~~~a> >

-100

-62-3 mV

E -0mV-100 v

-0 0

1

2

3

4 5 6 Time (min)

7

8

9

10

Fig. 5. Effects of suddenly changing [K]0 from 2 to 10 mm and from 10 to 2 mM on transmembrane potentials measured with K+-selective (a) and conventional (b) micro-electrodes. Membrane potential change measured with conventional micro-electrode is shown only in the reduction of [K]O.

Fig. 5 shows a similar experiment to those described above, using different [K]o at constant [Cl]0. In this experiment, the external solutions contained 2 and 10 mM-K instead of 5-4 and 30 mM-K of previous experiments. It was of interest to use the K concentration of 2 mm, because membrane potential deviated from the straight line for a K electrode in previous studies (Lee & Fozzard, 1975a). After a papillary muscle with diameter of 1'0 mm remained in 10 mm-K solution for about 30 min, as shown in the tracing b of Fig. 5, a resting membrane potential of - 62-3 mV was recorded. The membrane potential of - 62-3 mV is close to the average membrane potential of -61 *9 mV and the equilibrium potential ( -61 7 mV) calculated from the intracellular and extracellular K ionic activities which were obtained from similar muscles equilibrated in 10 mM-K solution (Lee & Fozzard, 1975a). On applying 2 mM-K solution, the membrane

673 K AND CI EFFECTS ON MEMBRANE VOLTAGE potential increased to - 90X1 mV and the full repolarization took about 3-2 min. The full repolarization time of 3X2 min was slightly faster than the 4 min required when 30 mM-K solution was suddenly replaced with 5-4 mm-K solution using the muscle with the same diameter. The membrane potential of - 90*1 mV is close to the average membrane potential of - 89*1 mV measured after equilibration in 2 mM-K solution, but deviated 5-4 mM

K CI

73 mm|

73 mm

-75-9 mV -72-6 mV

-78-2 mV

-100

K K

148 mM

148 mM

1

Ad-100

A

: > -50

5-4 mm 5_m

K

2

-

112

-SO E

-

6-0

5-2

10-2 .I

0

2

1

3

6

11-2 I

I

I

7

8

9

Time (min) -100

-77-8 mV -748 mV

-73-9 mV -70-5 mVY

E-50

-100

I Y'S

I

I

30

31

32 33 Time (min) Fig. 6. Effects on membrane potentials measured with conventional microelectrodes of suddenly reducing and restoring [Cl]o at constant [K]0. The values below the beginning and the end of each potential recording indicate the absolute value of the micro-electrode tip potential before impalement and after withdrawal. The membrane potentials above each recording were corrected for the tip potentials. 0

1

2

3

from the potential of the potassium electrode (Lee & Fozzard, 1975a). From the same muscle, a transmembrane potential of - 1507 mV was then measured with a K+-selective micro-electrode as shown in the tracing a of Fig. 5. On applying 10 mM-K solution the potential fell to - 123-9 mV and the full depolarization took about 1P2 min. On restoring 2 mM-K solution, the potential increased to -151-4 mV in similar time course to that in the tracing b of this Figure. The magnitudes of depolarization and repolarization (26-8 and 27-5 mV) are close to that (27.8 mV) of repolarization in the tracing b. These results indicate that there is no change in

674 H. A. FOZZARD AND C. 0. LEE intracellular K ionic activity during changes in the external K concentrations, and are consistent with the constant intracellular K ionic activity that we obtained from the papillary muscles equilibrated in different external K concentrations (Lee & Fozzard, 1975a). Variation of external Cl at constant external K The results of changes in [K]0 at constant [Cl]o suggest that in the heart muscle cells the chloride permeability is small relative to the K permeability. To test the suggestion and obtain the relative permeabilities of Cl and K, external Cl concentration was suddenly changed at constant [K]0 using solutions A and D in Table 1. Fig. 6 illustrates the effect of reducing and raising [Cl]o on the cell membrane potentials of a papillary muscle with diameter of 1-2 mm. After about 30 min equilibration of the muscle in Tyrode solution, a resting membrane potential of - 78-2 mV (left tracing of Fig. 6A) was registered. On applying 73 mM-Cl solution, membrane potential fell rapidly from - 78'2 to - 75-3 mV, and was maintained. After about 6 min in 73 mM-Cl solution, a membrane potential of - 72-6 mV was measured. Although the potential of - 72-6 mV is different from - 75.3 mV in the same solution, this difference of a few mV may be observed between individual impalements. On restoring 148-2 mM-Cl solution, the membrane potential increased rapidly only by about 3mV and was maintained. These potential changes between the on- and off-effects of 73 mM-Cl solution were symmetrical, as well as we could judge for such small changes. Similar results were obtained from the experiments shown in Fig. 6 B where the experimental conditions were identical to those in Fig. 6 A except the muscle remained in 73 mM-Cl solution for about 30 min. Similar experiments to those shown in Fig. 6 were done using the solutions of A and E (Table 1) with high Ca concentration of 3-6 mm because of the possibility that the propionate ion can interact with Ca ion, resulting in the activity change of Ca ion and consequent change in membrane potential. However, the results were similar to those obtained in the solutions with 1-8 mM-Ca concentration.

The results described above support strongly the view that Cl permeability is small relative to K permeability. Also the results are consistent with those observed in sheep Purkinje fibres (Carmeliet, 1961). When external Cl was replaced by acetylglycinate at constant external K, ro change in membrane potential was observed. However, the results are quite different from those observed in frog skeletal muscles (Hodgkin & Horowicz, 1959). In the single fibres of skeletal muscles, variation of [Cl]o at constant [K]o produced large transient potential changes, indicating that the cell membrane behaves as a Cl electrode. Fig. 7 illustrates the effect of reducing and raising [Cl]0 on the membrane potentials measured with K+-selective micro-electrodes in a papillary muscle with diameter of 1-3 mm. When Tyrode solution was suddenly

K AND Cl EFFECTS ON MEMBRANE VOLTAGE 675 replaced with a solution contained 73 mM-Cl, the membrane potential fell rapidly from - 110-9 to - 107-7 mV and was maintained (Fig. 7A). On restoring Tyrode solution after about 1 min, the potential returned rapidly to the original level. Fig. 7B and C are similar experiments to that in Fig. 7A except the muscle remained in 73 mm-Cl solution for about 30 min. The results shown in Fig. 7B and C are similar to those observed in Fig. 7A. These potential changes measured with K+-selective micro-electrodes (Fig. 7) are similar to those measured with conventional micro-electrodes (Fig. 6), and indicate that the intracellular K ionic activity does not change during alteration of [Cl]o. 5-4 mM

K

Cl

148

148

mm

mm

mV

-150

-110-9 mV

-100 -100

~

-107l7mV

-50 o

K

K

4

-111*1 mV

,

,

1

2

3

Time

(min)

mm

A

I

I

4

5

148 mm

Cl

73 mm

73 mm

-E1371 mV

-

,,

148 mM

F15

C

-142 8 mV

-140-1 mV

C

-134-0 mV

-10

l

mV -150

4

-0-iL50~.. I

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0

1

2

3

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,, E

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I

34 35 on transmembrane

Fig. 7. Effects of suddenly reducing and restoring [Cl], potentials measured with K+-selective micro-electrodes. Note that the magnitude and direction of potential changes are the same as those measured with conventional micro-electrodes shown in Fig. 6. It is possible that the small potential changes seen on changes of [Cli. was artifactual, caused by changes in the junction potentials of the agar bridge reference electrode saturated with KC1. Such artifacts from the reference electrode equilibrated for long time (40 min) in 200 mM-NaCl were reported when the external Cl concentration was reduced to 100 my (Strickholm, 1968). For this reason we sought to test whether the small potential changes in this study were due to such artifacts or not, even though the circuit system used in this study is different from that used by Strickholm. The system used in this test was identical to that used in the

H. A. FOZZARD AND C. 0. LEE

676

measurement of cell membrane potentials. The potential difference between the two calomel half cell without micro-electrodes was - 0-2 mV. Fig. 8A illustrates the effect of changing Cl on the potential measured between a micro-electrode and the agar bridge reference electrode, both immersed in 148 mM-Cl solution (Tyrode solution) for about 3 min before sudden application of 73 mm-Cl solution. The potential in 148 mM-Cl solution was - 22 mV which must be tip potential of the micro-electrode. On applying 73mM-Cl solution, the potential increased rapidly from -22 to -4-7 mV and then slowly drifted to a stable potential of - 3-1 mV. The rapid increase of the potential varied usually between 2 and 4 mV, depending on individual microelectrodes. The time required to reach a stable potential also varied from 1 to 3 min. -10 -148 mM-Cl 73 mM-Cl >

_5 _

148 mM-Cl

~~~~~~ ~ ~ ~Cl A

I_

-10

73

148

148 B

-5

_

Cl

0

E

0

50

E

148

148

73

Cl

r D

-1 E

-

I

0

I

I

I

I

I

2 6 3 4 5 Time (min) Fig. 8. Effects of sudden change in Cl concentration of Tyrode solution on the potential measured between an agar bridge reference electrode and conventional micro-electrode. See text for explanation. 1

The stable potential in 148 mM-Cl solution was different from that in 73 mM-Cl solution, as seen in the recording A. The differences were usually about 1 mV and might be due to the change in the tip potential. On restoring 148 mM-Cl solution, the potential decreased rapidly from - 3-1 mV to 0 and then drifted to a stable potential of - 1-8 mV with similar time course to that of previous potential drift on applying 73 mM-Cl solution. The recording B shows the potential changes in the same experimental condition as in A except the agar bridge reference electrode was equilibrated in 148 mM-Cl solution for about 1 hr before application of 73 mM-Cl solution. The

677

K AND Cl EFFECTS ON MEMBRANE VOLTAGE

potential changes in B are virtually identical to those measured between the same micro-electrode and the reference electrode equilibrated in 148 mM-Cl solution for a few minutes. The recording C shows the potential changes after the tip of the microelectrode used in B was broken. The potential changes were completely different from those observed in A and B. The recording D shows another example of similar result to those in A and B. In the recording D, the potential changes were measured between a micro-electrode with tip potential of - 9 mV and the agar reference electrode equilibrated in 143 mM-Cl solution for about 40 min. Eight micro-electrodes were tested and the results were similar. The results obtained in Fig. 8 indicate that small transient potential changes arise from the alteration of the tip potential of microelectrodes, but not from the junction potential of the reference electrode. The transient potential changes might not be seen during the measurement of cell membrane potentials since the micro-electrode tip was inside the cells and not in the solution. Therefore it may be concluded that the small changes of membrane potential on variation of [Cl]o (Fig. 6) are not artifacts but real membrane potential changes. On the other hand, the results suggest that the artifacts due to such small transient potential changes might occur when micro-electrodes are used as reference electrode. Finally it should be pointed out that the transient potential changes are different from those observed by Strickholm (1968) in their characteristic since the potential drifts were in opposite direction to those observed by him. 30 x 266

[K] x [Cl] -100 F E

J

-50

3

1

03 Time

2

3

4

5

(min)

Fig. 9. Effect on membrane potential of sudden change of solutions with a [K] x [Cl] of 800 (mM)2.

Variation of external K and Cl at constant product From the previous results in this study, it is expected that variation of external K and Cl concentration keeping a constant [K]o x [Cl]o results in similar membrane potential changes to those observed in variation of [K]0 at constant [Cl]0. To confirm this expectation, the potential changes were continuously measured during changes of external K and Cl concentration at the constant product of 800 (mM)2. For this experiment, solutions A and F of Table 1 were used. Fig. 9 illustrates the effect of sudden substitution of a solution containing 30 mM-K and 26-6 mM-Cl for Tyrode solution containing 5-4 mm-K and 148-2 mM-Cl in a papillary muscle with diameter of 0-9 mm. When the solution containing 30 mM-K and 26-6 mM-Cl was applied, the membrane potential fell from a resting potential of -78 to -39 mV. The full depolarization took about 1-2 min.

H. A. FOZZARD AND C. 0. LEE On restoring Tyrode solution after about 10 min equilibration in the solution containing 30 mM-K and 26-6 mM-Cl, the membrane potential increased from -38 to -75 mV. The full repolarization took about 3 min. The magnitudes and the time courses of the potential changes are similar to those observed for the changes of external K concentration at constant external Cl concentration. Thus the asymmetrical response of potential change was similar to that observed with change of [K]o at constant [Cl]0. These results are at variance with the observation in frog skeletal muscles (Hodgkin & Horowicz, 1959). In the single fibres of frog skeletal muscles, the membrane potentials changed rapidly so that full depolarization and repolarization took about 3 sec. But the results are consistent with the idea that the membrane of rabbit papillary muscle cells is primarily a K electrode. Relative permeability and conductance of K and Cl 678

The results described so far indicate that the cell membrane potential of the cardiac muscles is affected mainly by K ion and so K permeability is much greater than Cl permeability. In this section the relative permeability (PCJPK) and conductance (9C1I/K) of Cl and K will be calculated from the results obtained by the experiments described in the previous sections. To estimate the relative permeability with the data obtained in this study, it seems possible to use the method described by Strickholm & Wallin (1967) in which the relative permeability was calculated according to the following equation:

PCl/PK Tcl[K], fITK[Cl]o, =

(4)

where Tc, and TK are AVK/AVCI and AV /AVK respectively; [K]0 and [Cl]0 are external K and Cl concentrations; 6 is exp (F/RT VM). For AVl/IAVcl, AVTJ represents the sudden change in membrane potential when external Cl concentration was changed at constant external K concentration. This potential change could be obtained from the experiments shown in Fig. 6. AVc1 is (RTIF) In ([Cl]2I[Cl]l) where [C12 and [Cl]l are the two different external Cl concentrations used in the experiments. For AVMI/AVK, AViM represents the membrane potential change without altering the intracellular K ionic activity when the external K concentration was suddenly changed at constant external Cl concentration. This potential change could be obtained from the experiments shown in Figs. 2, 3 and 4. A/VK is (RT/F) In [K2/K1] where [K]2 and [K]l are the two different external K concentrations used in the experiments. At [K]o of 5 4 mm, Tc, value of 0 146 + 0008 (s.E.) was obtained from fourteen measurements and the resting membrane potential was - 76 1 + 1-4 mV (S.E.). At [Cl]o of 148-2 mm, TK value of 0915 ± 0 007 (S.E.) was obtained from twelve measurements. Using these results and eqn. (4), 0-11 for the relative permeability (Pcl/PK) was obtained. The relative permeability, about 01, is lower than that of about 0 5 for frog skeletal muscle cells (Hodgkin & Horowicz, 1959). Chloride (gcl) and K (9K) conductances were estimated according to the following equations: K=

'K/(VK-VK).

(6)

In eqn. (5) the driving force, V., - Vclwas taken as the potential difference between the equilibrium potentials of the external Cl concentrations used when the external

K AND Cl EFFECTS ON MEMBRANE VOLTAGE

679

Cl concentrations were suddenly changed at constant external K concentration. The potential difference calculated from equilibrium potential for 148 and 73 mM-Cl was 18-2 mV. ICI Cl current was estimated from the observed potential change and the membrane resistance of 10000 Q cm2 obtained by Weidmann (1970) in muscle. The observed potential change was 2-7 + 0 1 mV (s.E.) which was obtained from the experiments as shown in Fig. 6 for fourteen measurements. From these data, the chloride conductance, gcl appeared to be 0-015 mmho/cm2. Similarly the potassium conductance, gK was estimated to be 0-09 mmho/cm2. Thus the relative conductance gCl/gK was about 0-17. In the calculation of gcl and gK it was assumed that the membrane resistance is constant during the sudden changes of external Cl or K concentration. This is in agreement with the finding that the Cl component amounted to 10-30 % of the total membrane current (Deck & Trautwein, 1964). The relative conductance value of 0 17 may not be exact, since the membrane resistances are assumed to be constant, but they are probably changed by changing [Cl]. and [K]o. After reducing [Cl]o, the membrane resistance is little changed since the cell membrane was depolarized by a small value (Fig. 6), only 2-7 mV. After raising [K]o, however, the membrane potential was decreased by relatively large value to the K equilibrium potential (Figs. 2, 3 and 4). This depolarization might cause a drop in the membrane resistance as observed in sheep Purkinje fibres (Carmeliet, 1961). If the decrease in membrane resistance can be interpreted as a rise in gir then 9CI./9K would be smaller than 0-17. Thus the relative conductance value of 0-17 may be regarded as an upper limit in the membrane potential range observed in these experiments.

Asymmetrical response between rise and fall of external K Asymmetrical responses of membrane potential change between rise and fall in external K concentration are clearly seen in Fig. 2. In the muscle with a diameter of 1 mm (Fig. 2A), the full depolarization by a rise of [K]o from 5-4 to 30 mm took about 1-5 min and the full repolarization by a fall of [K]o from 30 to 5-4 mm took about 4 min. The half-time of the depolarization is about 5 sec, while the half-time of the repolarization is about 50 sec. These asymmetrical phenomena were also observed in the potential changes measured with K+-selective micro-electrodes as seen in Fig. 4. Similar asymmetrical phenomena were observed in the single fibres of frog skeletal muscle (Hodgkin & Horowicz, 1960). In order to explore the basis of the asymmetrical responses, calculations of some factors related to the responses are compared with the experimental results. In this analysis we make the following assumptions: (1) the papillary muscles used in this study are symmetrical circular cylinders, (2) the diffusion constant of K+ in the extracellular space of the muscles is the same as that in aqueous solutions with similar concentration, and (3) the cells from the surface of the muscle to the centre (axis) are electrically connected. When a K concentration, C1, in the solution around the muscle is suddenly changed to another concentration, CO, the K concentration, C, inside the muscle can be expressed by the following equation (Crank, 1956; Carslaw & Jaeger, 1959) 00 a-C-1 1-2n= J,(,8,/r)e =, 12 =

A7n

where x is a distance from the axis of the muscle, r is the radius of the muscle, J. and J, are Bessel functions of the first kind of order zero and first order, fi,,

680 H. A. FOZZARD AND C. 0. LEE (n = 1, 2 ...) are the positive roots of Jo(fJ) = 0, D is diffusion constant, and t

is time. The concentration profiles to be expected in the extracellular spaces of a cylindrical muscle bundle were calculated from eqn. (7) for various times after a concentration change. The calculation indicated that at the centre of a muscle of 1 mm diameter the K concentration would not change for 6 sec, and would obtain substantial equilibrium by 84 sec. 1-844 x 10J cm2/sec was used for D. In order to compare the time course of voltage change to that expected from a simple diffusion effect, the logarithmic relationship between the K gradient and potential must be taken into consideration. During the transient phase, some cells in the bundle will have different transmembrane K gradients. Presuming that the cell interiors are well connected electrically, the simplest method of calculation is to use the average K gradient, reflected in the average external K concentration, C. The quantity of K in the extracellular spaces at any time can be obtained by the product of eqn. (7) and 2irrldx and integration between 0 and r. or

Q= f27rrlCdx,

(8)

where I is the length of the muscle. From eqn. (8) the average K concentration is obtained by dividing Q by the volume of the muscle ffr2l. ¢ = Co -4

(CO-C,)

00

n=1

e

.f2Dt 2

(9)

This relation is illustrated in Fig. 10 for inward and outward diffusion. The average K concentration change in time is symmetrical. 98 % equilibrium by diffusion would be achieved in about 84 sec, presuming our assumption regarding the muscle geometry and K diffusion constant are correct. This value is in good agreement with the full depolarization time of about 1-5 min on increasing K from 5-4 to 30 mm in a 1 mm diameter fibre (Figs. 2A, 3B, 4A). However, repolarization took a much longer time (Figs. 2A and 4B).

Using the Nernst equation for a K electrode the time course of potential change can be plotted for comparison. The open circles in Fig. 11 are these calculations for the change between 5-4 and 30 mm and a comparable experiment in a 1 mm muscle. At resting potential the calculated potential was -79 mV, 3 mV more negative than the measured potential. This small difference may be ascribed to the deviation ofthe membrane from a K electrode at the concentration (Lee & Fozzard, 1975a). The time courses are quite close for depolarization; the half time was 5 sec and the time to full depolarization was 1-5 min, suggesting that the major determinant of the time course was indeed diffusion. However, when [K]0 was returned to 5*4 mm, repolarization of the muscle was much slower than that predicted by the simplified equations. An important factor not considered in this calculation is the rectifier property of the muscle membrane. In heart and skeletal muscle there is less resistance to K inward movement than K outward movement (inward rectification) (Katz, 1949; Hutter & Noble, 1960; Carmeliet, 1961). This relationship would favour slower repolarization, as illustrated by Naka.

K AND Cl EFFECTS ON MEMBRANE VOLTAGE

681

30

Inward diffusion 20 E 1: Pi

10

Outward diffusion

60

40

20

80

1

Time (sec)

Fig. 10. Average concentration. (C) of K ion in the extracellular space of a papillary muscle with diameter of 1 mm after external K concentration is suddenly changed from 5-4 to 30 mm (inward diffusion) and from 30 to 5.4 mm (outward diffusion). 30 mm

5-4 mm

K Ca

K

148 mm

-cI

mV .0

0

I

I-

0

0 0

I W

0

0

-70 -60 -50 -40 -30 -20 -10 I

I

I

I

I

0 20 40 60 80 100

A

I

I

IJ

I

I

I

0 20 40 60 80

I

100

120

140

I

-

160

I

_

180

I

_

200

I _

220

I

_

240

I

_

260

Time (sec)

Fig. 11. Relation between measured membrane potential (continuous tracing) and calculated membrane potential (open circles) after [K]o was suddenly raised and restored at constant [Cl]o. The continuous tracing (measured membrane potential) was a recording of membrane potential change obtained from a muscle with diameter of 1 mm. The open circles were calculated from differences between average K concentrations at various times. See text for explanation.

jima, Nakajima & Peachey (1973), but no attempt was made to calculate the influence of this factor because of complicated cell networks and insufficient information on the local currents that would flow under these conditions. On sudden reduction of [K]0 in normal fibres of frog skeletal

682 H. A. FOZZARD AND C. 0. LEE muscle, the slow repolarization was explained by the changes of membrane resistance concomitant with the changes of [K]0 which reflects a washout process of K+ from the T system. Brief exposures to high K solutions produced transient changes consistent with diffusion. Fig. 12 illustrates the differences in repolarization for fibres exposed to 30 mM-K for 3-64 sec. The repolarization times are 5-4 30

-100

-100

_________

50 :

S

5-4 mM -K

5-4 30

54 mM-K

-

A

J

B

-so

.-A~__

5-4 30

5-4 30

5-4 mM-K

5-4 mM-K

-100

-100 E

0C

-

L

.

/

1

-50

D

it O

-

54 mM-K 30

5 4 mm -K -100

-100E

E

-50

0 j 54 30 mM -K -100-

-50 0

54mM -K -100 FX

50 0

1

2

5 4 3 Time (min)

6

7

8

Fig. 12. Effects on membrane potential of sudden application and restoration of [K]0 with different periods applied to 30 mM-[K]0. The periods in A, B, a, D, E and F were 3, 4, 6, 12, 28 and 60 sec respectively. See text for discussion.

provided in Table 2. The change in time course of repolarization is no doubt due to the difference in distribution of K within the bundle at shorter times. On applying 30 mM-K solution in tracing A, the membrane potential of -75 mV fell rapidly to -58 mV. On restoring 5-4 mM-K solution after 3 sec exposure, the membrane repolarized in two phases, and the full repolarization took about 85 sec. In the tracing F of exposure duration of

K AND Cl EFFECTS ON MEMBRANE VOLTAGE 683 about 1 min, the membrane potential decreased from -74 to -47 mV and the full repolarization took about 260 sec. The full repolarization time as well as the fast repolarization rate after short exposure indicates that the membrane potential changes by altering [K]o are related to K diffusion process in the extracellular space of the muscle, and not to redistribution of ions across the cell membrane. Similar observations in single fibres of frog skeletal muscle was explained by K diffusion in T tubules of the fibre (Hodgkin & Horowicz, 1960). TABLE 2. Brief exposure to 30 mM-K

A B e D E F

Duration of exposure

Time of full repolarization

(sec)

(sec)

3 4 6 12 28 64

85 87 90 96 190

260

Variation of toxicity of external solution Fig. 13 illustrates the effect of suddenly reducing and restoring external tonicity on the membrane potentials measured with K+-selective and conventional micro-electrodes. When external isotonic Tyrode solution was replaced with 0-76 times hypotonic solution by using the solutions A and G in Table 1, the membrane potential measured with a K+-selective microelectrode increased from - 138-9 to - 141-9 mV as shown in Fig. 13A. On the other hand, the membrane potential measured with a conventional micro-electrode decreased from - 74-7 to - 69'5 mV, presumably due to a dilution of internal K (Adrian, 1956). After the muscle remained in the hypotonic solution for about 30 min, the isotonic solution was reapplied. On restoring the isotonic solution, as shown in the tracing a of Fig. 13B, the membrane potential measured with a K+-selective micro-electrode decreased from - 145-0 to - 141-6 mV and the potential measured with a conventional micro-electrode increased from - 71-9 to - 76-8 mV. The membrane potential changes between application and removal of the hypotonic solution were symmetrical even after the interval of about 30 min. Using eqn. (3), the intracellular K activities were calculated from the membrane potential changes. The intracellular K activities of 83-7 + 1-8 mm (S.E.) and 60-5 + 0-9 mM (S.E.) for the muscle in the isotonic and hypotonic solutions respectively were obtained from five measurements. The intracellular K activity of 60-5 mm is close to that of 63-6 mm, predicted from water movement across the cell membrane if the cells were an

H. A. FOZZARD AND C. 0. LEE

684

Isotonic T T~~~~~~~~~~

-141 9 mV +

-138 9 mV ;

A

-150

-100

-100 E

-50 1

1b

sog-5

i

I

I

I

I

I

I

I

0

1

2

3

4

5

6

E

Time (min)

Isotonic T Hoonc TT -141 6mV -1450mV -150

B

-150

-71-9 mV -76,8 mV

-10

-100

-50t:

t

E50

0

1

2

4 3 Time (min)

5

6

Fig. 13. The on- and off-effects of hypotonic solution (x 0 76) on membrane potentials measured with K+-selective micro-electrode (tracing a ofA and B) and conventional micro-electrode (b of A and B). e and T indicate impalements and withdrawals of the micro-electrodes. The potential changes measured with K+-selective and conventional micro-electrodes were opposite in their directions when external toxicity was suddenly changed.

osmometer, suggesting that the activity coefficient for intracellular K was little changed. Similarly the membrane potential changes were measured with K+selective and conventional micro-electrodes when external tonicity was raised and restored by using solutions A and H in Table 1. The directions of the potential changes were opposite to those of the potential changes in the hypotonic experiments. The intracellular K activities of 83-1 + 1-9 mM (S.E.) and 110-4 + 2-3 mM (S.E.) for the muscles in the isotonic and hypertonic solutions respectively were obtained from eight measurements.

K AND Cl EFFECTS ON MEMBRANE VOLTAGE 685 Fig. 14 shows these results (B) together with the results (A) obtained from the hypotonic experiments. The intracellular K activity of about 110 mm (right rectangle of B) is substantially lower than 125 mm (middle rectangle of B), the K activity predicted from water movement across cell membrane under the assumption of constant activity coefficient of intracellular K. Thus, the discrepancy between the measured and predicted values is about 15 mM. Hypotonic

Isotonic

Hypertonic

140

120 100 E 80

*60 40 20 AB

Fig. 14. The measured intracellular K+ activities (1) and calculated intracellular K+ activities (fl) in hypotonic, isotonic and hypertonic solutions. See text for explanation and S.E. of each value.

For explanation of this result in hypertonic solutions, one should consider the following possibilities. Firstly, when the hypertonic solution was applied the predicted amount of water may not have left the cells. In other words, the osmolarity of cytoplasm failed to increase 1P5 times, as in the external solution. However, this possibility is unlikely since cell water is reported to decrease to the predicted amount in this range of tonicity changes (Page & Storm, 1966). Water movement across the membrane would be much faster than solute movement. Further, Blinks (1965) found a linear relationship of fibre volume versus the reciprocal of osmotic strength over very wide range of osmotic strengths in frog skeletal muscle. Secondly, when the hypertonic solution was applied, a significant amount of K ions may have shifted out of the cytoplasmic compartment where the K activity was measured. In this case, the cytoplasmic K activity would be lower than that predicted by cell water movement. Thirdly, a change in activity coefficient of intracellular K ion may be involved in the discrepancy between the measured and predicted values. An increase in ionic strength itself would result in a decrease of activity

H. A. FOZZARD AND C. 0. LEE coefficient because of coulombic forces. If cytoplasmic K activity coefficient decreases with increases of K concentration like the mean activity coefficient of KCl in the range of 100-200 mm (Robinson & Stokes, 1965), the predicted intracellular K activity would be 118 mm instead of 125 mM (shown by dotted line in the middle rectangle of Fig. 14B). With this simple correction, the discrepancy would be 8 mm instead of 15 mm. However, the nature of the intracellular anion is not known, and the prediction by KCl solutions may be inaccurate. The change in intracellular K activity coefficient with K concentration would be more complicated, since the major intracellular anion is certainly not Cl ion. 686

DISCUSSION

It has generally been assumed that the cell membrane of resting cardiac ventricular muscle is permeable primarily to K, so that the resting potential is that of a K electrode. Our prior studies of intracellular K activity in heart muscle (Lee & Fozzard, 1975a) were interpreted as support for this assumption. However, since fast skeletal muscle cells may act as a Cl electrode (Hodgkin & Horowicz, 1959), we have sought to test the relative permeabilities of these two ions in heart muscle. The result of changes in [(K]0 with constant [Cl]. or of changes in [Cl]0 with constant [K]o substantiated the K electrode assumption, indicating that changes in [Cl]o influenced the membrane potential very little. Estimates of the ratio of PC1 to PK and gel to qg were made using the magnitude of voltage changes. While the measurements themselves were fairly accurate, the membrane voltage was not the same in both experiments. For this and other reasons the conductances of the two ions may not have been comparable in the two experiments, so that the calculated values should not be considered exact. Nevertheless, this problem does not jeopardize the significance of the relative conductance (gC1/9K) (see p. 679). In addition, conductances were calculated by use of Weidmann's measurement of membrane resistance for ventricular muscle of 10 kQ cm2. Since the membrane surface area he used was an approximation, these calculations of partial membrane conductances are also approximate. No effort should be made to compare these conductance values to those of Deck & Trautwein (1964), in Purkinje fibres, since they also had no accurate method of measuring membrane surface area. On the other hand, the conclusion that PC1 is much smaller than PK is obvious from the voltage responses to ion changes, and is not endangered by problems with surface area estimation. If indeed gc is quite low, the influence of Cl ions would be small. However, during the plateau of the Purkinje action potential, there may be a

K AND Cl EFFECTS ON MEMBRANE VOLTAGE 687 rise in gc1 (Dudel et al. 1967; Fozzard & Hiraoka, 1973), associated with a fall in gE because of inward rectification. Therefore, it may be suspected that the active cell membrane is more susceptible to changes in Cl than the resting membrane. The time course of the membrane potential change was fast on depolarization and slow on repolarization. Part of this asymmetry could be accounted for by diffusion of K into the unstirred extracellular space of the muscle bundle. For these calculations, the self-diffusion coefficient for K was used, since there was no reason for hindrance to K movement in the large extracellular spaces. Repolarization could have been fitted by adjusting the diffusion coefficient to a lower value, but a difference would then exist for depolarization. This implies that the asymmetry cannot be explained by a lower K diffusion coefficient in the extracellular space. If the K diffusion coefficient used in our calculations is the true value, the difference between it and the value calculated by Nakajima et al. (1975) may be attributed to a difference of the K diffusion coefficient in the cardiac muscle extracellular space and in the T tubules of skeletal muscle. They reported that the apparent diffusion constant of K+ was substantially lower in the T system than that in an aqueous solution. In heart muscle the T tubules are relatively scarce and of large diameter. The remaining asymmetry of response of K change was in the direction to be expected for K inward rectification. Measurement of intracellular K activity during solution changes provided direct evidence that [K]i was not altered. This conclusion is necessary for the calculation of permeabilities, and further supports the earlier findings of Lee & Fozzard (1975a). If KCl did enter the fibres, it was accompanied by water sufficient to prevent a rise in intracellular K activity. Studies of Carmeliet & Janse (1965) suggested that [Cl]1 did gradually rise when [K]0 was increased, but without an increase in cell volume. Our measurements seem in conflict with their conclusion, since an increase in KCl content without swelling would have increased intracellular K activity. An additional observation from the simultaneous measurements with K+-selective micro-electrodes is that the electrodes faithfully followed the time course and magnitude of voltage change measured by conventional pipettes. This result strengthens the impression that the K-selective electrodes do not injure the cell sufficiently to produce a large shunt at the impalement site. Further the response times of these higher resistance electrodes was rapid enough to follow membrane potential accurately. Studies with changes in [K]i by alteration of solution tonicity were difficult to interpret. While hypotonic solutions produced the expected change in intracellular K activity within a few millimoles, hypertonic solutions produced much less change. Under these conditions, the cells 27

PHY 256

688 H. A. FOZZARD AND C. 0. LEE may not have behaved as an ideal osmometer because of mechanical constraints to shrinkage. However, as discussed previously (Akiyama & Fozzard, 1975), the intracellular anion is not Cl, but a mixture of polyvalent organic and inorganic anions. Under these conditions, the intracellular ion activity coefficient might decrease substantially with increasing concentrations. Supported in part by USPHS grants No. HL 11665 and No. HL 05673. We appreciate the assistance of Mr Harold Alexander in certain technical aspects of these experiments. REFERENCES ADRIAN, R. H. (1956). The effect of internal and external potassium concentration on the membrane potential of frog muscle. J. Physiol. 133, 631-658. ArIYAmA, T. & FoZZARD, H. A. (1975). Influence of K ion and osmolality on the resting membrane potential of rabbit ventricular papillary muscle. Circulation Rem. 37, 621-629. BLiNKs, J. R. (1965). Influence of osmotic strength on cross-section and volume of isolated single muscle fibres. J. Phy8iol. 177, 42-57. CAuXIEMT, E. E. (1961). Chloride ions and the membrane potential of Purkinje fibres. J. Physiol. 156, 375-388. CARMELET, E. E. & JANSE, M. (1965). Intracellular chloride concentration in cat papillary muscles. Influence of external K concentration. Arch8 int. Physiol. Biochim. 73, 174-175. CARSLAW,H. S. & JAEGER, J. C. (1959). Conduction of Heat in Solids, 2nd edn., p. 199. Oxford: Clarendon. CRANK, J. (1956). The Mathmatic8 of Diffusion, p. 66. Oxford: Clarendon. DECK, K. A. & TRAuTWEIN, W. (1964). Ionic currents in cardiac excitation. Pflugers Arch. gee. Physiol. 280, 63-80. DUDEL, J., PEPER, K., RMDEL, R. & TRAUTWEIN, W. (1967). The dynamic chloride component of membrane current in cardiac Purkinje fibres. Pflusgers Arch. ge8. Physiol. 295, 197-212. FOzzARD, H. A. & HIRAoKA, M. (1973). The positive dynamic current and its inactivation properties in cardiac Purkinje fibres. J. Physiol. 234, 569-586. GIBBONS, W. R. & FOZZARD, H. A. (1971). High potassium and low sodium contractures in sheep cardiac muscle. J. gen. Physiol. 58, 483-510. HODGKIN, A. L. & HOROWICZ, P. (1959). The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Physiol. 148, 127-160. HODGKIN, A. L. & HoROwIcz, P. (1960). The effect of sudden changes in ionic concentrations on the membrane potential of single muscle fibres. J. Physiol. 153, 370-385. HUTTER, 0. F. & NOBLE, D. (1960). Rectifying properties of cardiac muscle. Nature, Lond. 188, 495. HUTTER, 0. F. & NOBLE, D. (1961). Anion conductance of cardiac muscle. J. Physiol. 157, 335-350. KATZ, B. (1949). Les constantes 6lectriques de la membrane du muscle. Arch2 Sci. physiol. 3, 285-300. LEE, C. 0. & ARMSTRONG, W. M. (1974). State and distribution of potassium and sodium ions in frog skeletal muscle. J. membrane Biol. 15, 331-362. LEE, C. 0. & FOZZARD, H. A. (1974). Electrochemical properties of hydrated cationselective glass membrane: a model of K+ and Na+ transport. Biophys. J. 14, 46-68.

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