«THE RUBIDIUM AND POTASSIUM PERMEABILITY OF FROG MUSCLE MEMBRANE BY R. H. ADRIAN From the Physiological Laboratory, University of Cambridge (Received ...»
J. Phy8iol. (1964), 175, pp. 134-159
With 13 text-figureM
Printed in Great Britain
THE RUBIDIUM AND POTASSIUM PERMEABILITY OF
FROG MUSCLE MEMBRANE
BY R. H. ADRIAN
From the Physiological Laboratory, University of Cambridge
(Received 19 May 1964)
Rubidium is known to have effects on excitable tissues which are in
many ways similar to the effects of potassium. Rubidium in the external
solution depolarizes the membrane, though whether more or less effectively than potassium depends on the tissue. In frog nerve and muscle rubidium is less effective than potassium, and in frog muscle at relatively low external concentrations ( 20 mM) about twice as much rubidium as potassium is needed to produce equal depolarizations (Feng & Liu, 1949;
Sandow & Mandel, 1951; Sjodin, 1959). Substantial amounts of the intracellular potassium of rat muscle can be replaced in vivo by rubidium (Relman, Lambie, Burrows & Roy, 1957) and virtually complete replace- ment in vitro has been reported by Lubin & Schneider (1957) for frog muscle. It seemed possible to investigate the electrical propertles of frog muscle after replacing internal as well as external potassium by rubidium, and to consider all the four possible combinations of internal and external potassium and rubidium.
Adrian & Freygang (1962a, b) have suggested that there are two, spatially separated, pathways for the movement of potassium across the membrane of striated muscle. They considered that the anomalous rectification, characteristic of fast striated frog fibres (Katz, 1949), might take place in a membrane separating the sarcoplasm from a part of the endoplasmic reticulum which is connected to the extracellular fluid by a low electrical resistance. The behaviour of the conductance in solutions with a low potassium concentration (2.5 mM) could be explained by the division of the potassium current between two pathways; a reticular pathway allowing large inward but only very small outward currents, and a second pathway with a voltage-dependent potassium conductance similar to other excitable tissues. It was suggested that the surface of the fibre was permeable to chloride and was probably the site of the perme- ability changes responsible for the action potential.
The experiments reported in this paper were done to compare the move- ments of potassium and rubidium in the two postulated pathways. If Rb AND K IN MUSCLE 135 the properties ascribed to the separate pathways could be shown to depend on the presence of rubidium and potassium in different ways, the case for postulating separate mechanisms would be strengthened. A preliminary account of this work was presented to a meeting of the Physiological Society (Adrian, 1963).
METHODS All measurements were done on sartorius muscles of English frogs (Rana temporaria) at room temperature (18-23' C). The method for measuring the membrane current and potential, and its theoretical basis have been described in detail by Adrian & Freygang (1962a). Three electrodes are inserted into the same muscle fibre in line so that each electrode is 445,c from the next electrode and the last electrode is 445,u from the end of the fibre. The change in membrane potential (Va) produced by a current pulse delivered through the electrode furthest from the end of the fibre is measured by the electrode nearest the end of the fibre. The two electrodes near the end of the fibre measure the difference in potential (Vb - Va) at 445,u (a) and 890,u (b) from the end of the fibre. (Vb - Va) is the voltage drop along the intemal longitudinal resistance of 445,u of fibre, and is approximately proportional to the membrane current altering the membrane potential near the end of the fibre. The membrane conductance is very nearly directly proportional to (Vb - Va)/Va.
When the membrane resistance is non-linear (Vb - Va)/Va is a good approximation to the chord conductance at Va as long as (Vb - Va)/Va is less than about 0-15.
The experimental results are plotted in two different ways. (Vb - Va), abbreviated to AV, can be plotted against the internal potential of the fibre (the internal potential of the fibre in the absence of current plus Va) to give the relation between membrane current and membrane potential. The convention adopted makes positive values of AV represent outward membrane current. Alternatively (Vb - Va) x 100/Va, abbreviated to AV/V %, can be plotted against the internal potential to give the relation between membrane conductance and membrane potential. For an 80je fibre with an intemal specific resistance of 250 Q cm, AV = 1 mV is equivalent to a membrane current of 2-7 x 10-6 A/cm2, and AV/V = 1 % is equivalent to a membrane chord conductance of 2-7 x 10-5 mho/Cm2.
In the majority of the experiments the characteristics of any particular fibre were measured in one solution only. In a few, however, the solution was changed with the three electrodes in the fibre. The muscle was placed in a groove with a cross-sectional area of 05 cm2. Flows along the groove of 15-20 ml./min could be tolerated; higher flows caused electrode movement and damage to the fibre.
Resting and action potentials were measured with micro-electrodes by conventional methods. The resting potential is given as the inside potential minus the outside potential, and the action potential as the change in potential from the resting level.
Solution&. The solutions used in these experiments were based on the solutions described by Hodgkin & Horowicz (1959). Their compositions are shown in Table 1. Solution A is a Ringer's solution of conventional composition; B is similar but contains 2-5 m-equiv/l.
Rb+ in place of K+. Solution C is isotonic with A, contains 2-5 m-equiv/l. Rb +, but no chloride. Solutions D and E are isotonic with A, contain no chloride, and 100 m-equiv/l.
K+ and Rb +, respectively. Solution J contains 100 m-equiv/1. K+, 10 m-equiv/1. Rb+, but no chloride.
Two solutions were used for replacing internal potassium by rubidium. Solution K is the same as the solution used by Lubin & Schneider (1957) except that it contains, in addition to the constituents in Table 1, 2-5 mg/l. rather than 50 mg/l. tetracycline hydrochloride.
Solution L is a sulphate solution with 100 m-equiv/l. Rb + and a small quantity of glucose.
It contains no antibiotic. Solution M is the potassium analogue of solution L.
When soaking for long periods muscles were attached to a glass frame by threads tied to R. H. ADRIAN 136
0~~~~~~~ 00 Rb AND K IN MUSCLE 137 their tibial tendons and pelvic bones. Four muscles on a frame were suspended in a flask containing 100 ml. of solution, and the flask was immersed in a water-bath whose temperature could be controlled. The solution was bubbled with the appropriate gas mixture (Table 1).
Estimation of intracellular cations. Muscles were prepared for analysis by drying to constant weight. The dried muscles were put into Pyrex volumetric flasks and extracted with a hot CsCl solution (900 C in a drying oven) for several hours. The flask was chosen to give a total concentration of the cations extracted from the muscle of about 250,u-equiv/l.
Extraction of dried muscles with hot water leaves neither sodium nor potassium bound to the muscle protein (Adrian, 1960); CsCl solution appears just as effective. Rubidium, potassium, and sodium were estimated with a Zeiss PQM II flame spectrophotometer with glass prisms in the double monochromator. With solutions without rubidium, and containing 250 /z-equiv/l. K +, no light was detectable at the rubidium emission line (780,u). With solutions containing 250,u-equiv/l. Rb +, the light detected at the potassium emission line (768,u) was less than 1 % of the light detected at 780,u. This probably represents potassium present in the rubidium salts rather than a failure to discriminate light of the two wavelengths. No correction was made.
To reduce the interferences in the flame between potassium and rubidium when both are present in the same solution, all solutions for flame-photometric estimations included 200 p.p.m. (2.38 mm) CsCl. The presence of the CsCl enhanced the emission of both potassium and rubidium by about 30 % and made the emission of a constant concentration of rubidium independent of the potassium concentration and vice versa. Muscle extracts were compared with two standard solutions whose compositions are given in Table 2. For potassium and rubidium the emissions of S2 were twice those of S1 and linear interpolation was used to find the concentrations in the unknown solutions. For sodium the emission of S2 was slightly less than twice the emission of S1, but since the sodium concentration of the muscle extracts seldom exceeded 50,u-equiv/l. this non-linearity was not taken into account. A small increase in accuracy could probably have been achieved by a large increase in the number of standard solutions used for comparison. The over-all accuracy is probably no better than about 5 %. The intracellular cation concentrations are expressed as m-mole/kg fibre water.
In calculating the quantities of intracellular ions and the fibre water, the extracellular space was assumed to be 12-5 % of the muscle wet weight (Desmedt, 1953).
RESULTS Replacement of intracellular potassium by rubidium. The method of Lubin & Schneider (1957) was used initially to prepare muscles with rubidium as the principal internal cation. Muscles were soaked at 250 C for 48 hr in solution K (Table 1). Virtually total replacement was produced by this procedure but these muscles behaved as if they contained considerable quantities of chloride. Lubin & Schneider found that the membrane potential of their Rb-containing muscles was sensitive to the replacement of external chloride by sulphate, and that these muscles failed to repolarize when the external rubidium concentration was lowered.
Their Rb-containing muscles did not respond to electrical excitation with an action potential. It seemed therefore essential to arrange the soaking solution so that there was little or no chloride in the muscles at the end of the soaking period. Muscles were soaked at 90 C in a solution which contained sulphate as anion and 100 m-equiv/l. Rb+. The solution R. H. ADRIAN 138 contained 1O m-mole/l. glucose and was bubbled with 100 % oxygen.
Figure 1 shows the time course of the replacement of the internal potassium by rubidium in this solution (L, Table 1). Replacement of all but about 20 m-mole/kg fibre water takes place in 5 days along an approximately exponential time course with a half-time of one day. During this time the sodium content of these muscles does not rise. Muscles remain translucent in this solution and at the end of 5 days show only an occasional clotted fibre. The mean internal cation concentrations ( S.E. of mean) of sixteen 140 V 120 \
muscles soaked for between 94 and 120 hr in the 100 m-equiv/l. Rb + sulphate solution were: K+, 23 + 2 m-mole/kg fibre water; Rb+, 125 + 2 m-mole/kg fibre water; Na+, 8 + 1 m-mole/kg fibre water. Four of the sixteen muscles were estimated directly from the soaking solution (L) and the rest after up to 1 hr in a Ringer's fluid made with RbCl (B). There was no significant difference between the two groups. The total cation concentration is very close to that of fresh sartorius muscles estimated in the same way and expressed in the same units (K+, 139 m-mole/kg fibre water;
Na+, 15 m-mole/kg fibre water).
Though sulphate is generally regarded as an impermeant ion, it is possible that a detectable entry might occur in the course of 5 days. An entry of sulphate would cause a swelling of the muscle and in consequence a fall in the dry-to-wet weight ratio. The mean dry-to-wet weight ratio of the sixteen muscles soaked for between 94 and 120 hr in the 100 m-equiv/l.
Rb + sulphate solution was 20 3 + 0 4 %. The mean dry-to-wet weight ratio of fresh muscles Rb AND K IN MUSCLE 139 prepared by the same method is 21F5 + 0-15 % (+s.E. of means, Adrian, 1960). If the difference in the dry-to-wet weight ratio is all due to swelling of the muscle, it represents only a 6 % swelling and a sulphate entry of about 5 m-mole/kg fibre water.
If the half-time for the loss of potassium into the rubidium solution is taken as 24 hr, the initial outward movement of potassium is only 2-3 pmole/cm2/sec (calculated assuming a fibre diameter of 80,u). The net membrane current is zero. Assuming that the contribution of sodium to the current is negligible, the rubidium influx is equal to the potassium loss.
The membrane potential at the beginning of the soaking period can therefore be used to estimate the relative permeabilities of potassium and rubidium by means of the following equation.
E = RT PRb[Rb]o Tn PK[K]l F When a muscle, which has been in a sulphate solution with 100 m-equiv/l.
K+ (solution M) for long enough to lose its internal chloride (1 hr), is put into the 100 m-equiv/l. Rb+ sulphate solution (L) the internal potential is - 23-5 + 0 4 mV (19 fibres, 4 muscles; ± S.E. of mean). Neglecting any effect of sodium this potential suggests that the replacement of potassium by rubidium takes place across a barrier which behaves as if it were 1-8 times as permeable to potassium as to rubidium. At the end of the 5 days' soaking, when there is rubidium on both sides of the membrane the internal potential is -10 + 0 3 mV (38 fibres, 5 muscles; ± S.E. of mean).
The potential to be expected with only rubidium on both sides of the membrane, assuming an internal concentration of 140 m-mole/kg fibre water and equal activity coefficients, is - 8-5 mV. Making allowance for the fact that 20 m-mole/kg fibre water of the internal cation are potassium, a value of 1-8 for the ratio of permeabilities makes the expected internal potential -11 mV.
Figure 2 (filled symbols) shows an experiment where three pairs of muscles were soaked in the 100 m-equiv/l. K+ sulphate solution (M) for 48 hr. Three were analysed from this solution and the remaining three were soaked for 6, 24, and 48 hr respectively in the 100 m-equiv/l. Rb + sulphate solution. The time course for the replacement of potassium by rubidium is the same as in freshly dissected muscles, shown in Fig. 1. However, muscles soaked for 96 hr in the 100 m-equiv/l. K+ sulphate solution appear to survive much less well than the muscles in the 100 m-equiv/l.
Rb + sulphate solution. They show a marked decrease in the dry-to-wet weight ratio, and the internal sodium rises at the expense of potassium.