An electrogenic component in the membrane potential of Tetrahymena

Conzp. Biochem. Physiol. Vol. 7lA, No. 2, pp. 335-344, 1984 Printed in Great Britain

c

0300-9629/84 $3.00 + 0.00 1984 Pergamon Press Ltd

AN ELECTROGENIC COMPONENT IN THE MEMBRANE POTENTIAL OF TETRAHYMENA J. G, CONNOLLY and G. A. KERKUT Department of Neurophysiology, School of Biochemical and Physiological Sciences, Southampton University, Southampton SO9 5NH, UK (Tel 0703 559122) (Received 23 May 1983) Abstract-l. The average resting potential of Terrffhymena vorax was - 24.15 f 0.78 mV (n = 139). 2. The addition of Ouabain at 10s5 M resulted in a decrease in the membrane potential of approximately 30”/,. 3. The response of the resting potential to changes in temperature was measured to determine whether it was dependent upon an active component. The response of the membrane potential to cooling was slower than the response to warming, although there was no significant difference in overall magnitude between the two types of response. 4. The Q10 value for the membrane potential changes in control solution (Na +-free) was 1.22 + 0.03 (n = 25). This is significantly greater (P < 0.01) than the value predicted by electrodiffusion theory (1.035)and suggests that there is some Na+ independent active transport involved in the maintenance of the membrane potential. 5. In control solution containing 20 mM Na’ the Q,a value was 1.44 + 0.6 (n = 16).This is significantly greater (P < O.M)l) than the response in control solution alone, indicating that the Na+ ion may be involved in an electrogenic transport mechanism. 6. Ouabain significantly (P < 0.01) reduced the Q,. value in Na+ solution from 1.44 k 0.06 (n = 16) to 1.09 rt 0.01 (n = 6). The reduction ofthe Qr,, value in control solution (Na+-free) from 1.22 + 0.03 (n = 25) to 1.14 + 0.01 (n = 6) was not significant at the 1% level (0.25 > P > 0.1). This suggests that an electrogenic Na* --K+ ATPase contributes to the membrane potential in Tetrahymena. The hyperpoia~sing effect of this ion pump may stabilise behaviour by reducing the number of spontaneous regenerative depola~sations. 7. Screening of localised electrical events by high resistance pathways in Tetrahymena and Paramecium is discussed. These pathways may be mediated by specialised membranes with reduced permeability so that there is considerable electrical heterogeneity among the membranes of the cell. Thus it is suggested that membrane constants obtained for Tetrahymena, and possibly mammalian neurones also, should not be considered as the product of simple isopotential systems. but rather as the complex average of many electrically distinct components.

INTRODUCTION Active transport of solutes may be classified as a primary or secondary process. Primary active transport involves the coupling of the passage of solutes across the membrane to the advancement of a chemical reaction. Examples of this type of “pump” are the Na+-K+, Mgzf activated ATPase (Skou, 1957) and the CaZ’-ATPase of the sarcoplasmic reticulum (Hasselbach and Mackinose, 1963). Secondary active transport involves the exchange of two solutes. One of these flows down its concentration gradient and drives the other against its gradient. The Na+-Ca” exchange in cardiac muscle (Reuter and Seitz, 1968) typifies this class of pump. The number and charge of ions exchanged across the membrane may not be equal. If the net charge transferred across the membrane is zero, the pump is said to be electroneutral, if not, the pump is called electrogenic. An e~ectroneutral pump can indirectly create a resting potential because of the selective permeability of the membrane to different ions (Goldman, 1943; Hodgkin and Katz, 1949) and the presence within the cell of non-ditfusible anions (Donnan, 1911). However an electrogenic pump can make a further contribution to the membrane potential by directly separating charges across the membrane. If the flow of charge is small then the immediate

contribution of the pump will be small and its effect will be noticed as a long term change in ion concentrations across the membrane. Such is the case in the squid axon (Hodgkin, 1951), and the behaviour of the membrane potential in this cell is successfully described by electrodiffusion theory (Goldman, 1943 ; Hodgkin and Katz, 1949 ; Hodgkin and Huxley, 1952a, b; Hodgkin et al., 1952). However, if the current flow across the resistance of the membrane is at any given instant larger, it may become significant enough to make a direct and immediate contribution to the membrane potential. Electrogenic pumps of this type have been described in several preparations, including Helix aspersa (Kerkut and Thomas, 1965), Anisadoris (Marmor and Gorman, 1970), Acerabularia (Saddler, 1970), heart (Deleze, 1960) and mitochondria (Mitchell, 1961). A membrane potential which includes an immediate electrogenic component will respond differently to changes in temperature (Ling and Gerard, 1949; Kerkut and Ridge, 1961) when compared with one which is essentially the result of simple diffusion (Goldman, 1943 ; Hodgkin, 1951). An electrogenic component in a membrane potential would result in a greater potential response to changes in tem~rature than would be expected from calculations based on electrodiffusion theory. The proportional change in 33.5

J. G. CONNOLLY and G. A. KEKK;III

336 Thermocouple Heat exe hanaer

/waterin

Suction electrode

/

reading was displayed on a temperature meter and rccol-dud on a Watanabe pen recorder. The temperature sensitivity of the electrodes was measured before and after recording from the cell. Any electrodes which showed a potential drift of greater than kO.5 mV per 10 C temperature change were rejected. The temperature measuring system was accurate to within iO.2 C within the temperature range investigated f 13 27, C). Voltage measurements were made using a digital voltmeter and recorded on a Watanabe pen recorder. but because of the lescl of background potential fluctuations in Tetrahymena, are probably only accurate to i 0.25 mV. Treatment

of re.su1t.s

01 Qlo. The Q,, value describes the proportional change in the magnitude of a parameter for a IO C change in temperature. If membrane potential is a function of the absolute temperature(T) only, then the ratio of the values of potential at two different temperatures will equal the ratio of the absolute value of those temperatures. Considering a 10°C change about 19‘C (292 K. approximately the average temperature at which the experiments were performed) then the temperature ratio (Q,,, value) = 297j287 = 1.035. This Qlo value would then be expected to represent the ratio of the membrane potentials at 297 and 287 K for a non-electrogenic resting potential. Most experiments did not span exactly the 1O’C between 14 and 24: C. For all ceils, a value for the change in mV/ C wa\ first calculated. This result was then extrapolated about the average potential to give a Qin value. An example is given Calculation

-.

-.__

Light SO”RX

Fig. 1.Schematic diagram of apparatus used for recording the resting potential of Tetrahymena.

membrane potential for a 10°C change in temperature can thus be used as an indication of the dependence of a resting potential upon metabolism and electrogenic components (Lund and Moorman, 1931; Kerkut and York, 1969). The present work defines the normal range ofresting potentials for ~efru~y~e~u norax. The Qlo value for membrane potential in control solution and in the presence of Ouabain and/or Na+ is determined in order to investigate whether there is an immediate, active component in the resting potential of Tetrahymena. MATERIAL

below :

T,: 17’C. T,: 23’C, 7: 20 C. mV,: 23.5. mV,: 26.5. I%\‘: - 25, ArnV~A~ = 0.5 mV ‘Cm ‘, Thus there is a change of 0.5 mV for a 1 ‘C change in temperature. This is then extrapolated to a 5 mV change in resting potential for a IO’C change in temperature about -25 mV, which gives a Qlo value of 27.5122.5 = 1.22. Where appropriate, the Student’s t-tests for paired and independent samples were used to determine the significance of the results.

AND METHODS

Intracellular recordings were obtained from Tetrahymena uorux which had been grown axenically and harvested at 20°C as described previously (Connolly and Kerkut, 1981). The apparatus used for recording from the cells is shown in Fig. 1,The Perspex bath normally contained control solution (1 mM KCI, I mM CaCI,, 0.t mM EDTA and I mM Tris-.HCI pH 6.8). A 2 cm3 syringe was used to introduce different solutions into the bath through a narrow inlet. Gentle, steady pressure would ensure that the flow ofsolution directed towards the preparation by the baffle did not release the cell and did not exceed the capacity of the constant head device behind the prism to remove the solution. When methylene blue dye was added to the bath it was found that the bath volume of 1.25cm: was completely cleared with 5

sec. The preparation was illuminated from the base of the bath and viewed through a stereoscopic microscope mounted in the same plane as the observer in Fig. 1.This elevation gives dark field illumination and facilitates 3-dimensional

RESULTS

Control

resting potential

The average resting potential of 139 cells in control solution was - 24.15 f 0.78 mV. The range was from -10 to -44 mV if cells undergoing regenerative hyperpolar~sations were excluded from the sample. A histogram depicting the frequency distribution of resting potential is provided in Fig. 2. The average input resistance of 26 cells in control solution was 47.8 ): 2.9 MR. The effect ofO&ain

on the membrane potrntiuf

The effects of Ouabain (Strophanthin-G) addition to cells in control solution is shown in Table 1. In all but 40

perception. The temperature of the bathing solution was altered by passing hot or cold solutions through two glass tubes located in the bath which acted as heat exchangers. Temperature measurements were made using a thermocouple placed in the bathing solution and adjacent to the cell. The temperature Abbreviations

[X ‘Ii refers to the intracellular concentration of species X+. [X+1, refers to the extracellular concentration. C-V is the contractile vacuole. “Na” solution” refers to controi solution containing an added 20 mM NaCI.

‘5

10

15

20

25

W

Resting potenttal Fig. 2. A frequency

35

40

45

( - mV 1

chart of resting potentials 1:0f
of ~~~~~~~~~~~~~~~~~

331

Tetrahymena membrane potential Table 1. The effect of Ouabain

addition

on resting potential

Response

Ouabain

M

lo-’ 10-6 lo-~ 5 x 10-S 10-b

time

R.P.,

R.P.,

(set)

(mV)

(mV)

90 50* 10 94 + 9.3 66.7 * 14.5 125

-18 -25 -21.5 -24.7 -25

-15 * 3.4 f 1.7

-16k 1 - 18.2 i 3.2 -18.3k4.4 - 10

one cell there was a gradual depolarisation which took an average time of 82 k 8 set (n = 12) to attain the maximum response. An example of such a response is shown in Fig. 3A. In one instance at 10m6 M there was no apparent response to Ouabain. The maximum response at lo- ’ M may not be complete whereas the maximum response at 10m4 M probably involves a non-specific component. Attempts to reverse completely the effects of Ouabain were unsuccessful unless washing followed almost immediately. In the results quoted in Table 1 washing was carried out 30 set after addition of the Ouabain. If this was not done the cells would continue to depolarise at a reduced rate and eventually become unviable. The average input resistance of the cells in 10m5 M Ouabain as 45 + 14.1 MR (n = 5). This is not significantly different from the input resistance in control solution alone (P > 0.6) which is 47.8 f 2.9 Mn (n = 26). Thus it would seem that Ouabain added in this way is not causing a non-specific reduction in membrane resistance. The average y0 change in resting potential after addition of 10m5 M and 5 x 10m5 M Ouabain was 31.9 + 4.3% (n = 8). This may be an approximate indication of the contribution of electrogenic pumping by a Na+-K+ATPase to the resting potential of these cells. Resting potential changes in control solution Figure 4 shows examples of the response of the resting potential of Tetrahymena to temperature change. This response typically followed temperature o

0 2 80 .

(a) Cortrol Ouabav

-10

AmV +3 +9* I +9.3 * 1.1 +6.4 + 1.9 +15

:/,change

n

16.7 36.0 i 4 34.8 + 4.6 27.lk8.9 60.0

1 2 5 3 I

changes with a time lag of 5-10 sec. Figures 4(a) and (b) contrast the range of the responses. Figures 4(a) and (c) demonstrate that the responses were reversible, with no significant differences between paired cooling and warming responses in individual cells or between the independent cooling and warming responses given in Table 2. It is interesting to note, as Kerkut and Ridge (1961) observed in crab, insect and frog muscles, that the resting potential is slower to react to cooling than to warming. This raised the question as to whether it was reasonable to extrapolate individual results to give a Qlo for the whole temperature range. This assumes that the rate of change of resting potential for a 1°C change in temperature is constant over the range of temperature studied. Responses in Table 2 whose mean temperature during the experiment was greater than the mean of the whole group (18.7”C) were compared with those responses whose mean temperature was lower than the group mean. There was found to be no significant difference between these groups. The assumption therefore seems justified for these cells over the temperature range studied. The average change in resting potential for a 10°C change in temperature about 18.7”C was 4.2 + 0.06 mV (n = 25). This is significantly greater than the value 0.76 mV which would be predicted by the Nernst equation (P < 0.01). The experimental Q,,, value of 1.22 & 0.03 is also significantly larger than the theoretical value (P < 0.01). These are strong indications of a metabolic contribution to the membrane potential of Tetrahymena uorax.

solution

-14

1O-5 M

a> F

IO set

-20 -30 t -24

5

3

H

2 K

(b)

z

(c) Na solution + 10m5M Oua barn

Nat so\utlon

"r

F

26°C

G 25

e L

25°C 14°C

15

Fig. 3. The effects of Ouabain

addition

on the resting potential

and its response

to temperature

changes

190°C 13 5”C/

Fig. 4. Examples of resting potential responses to temperature

ch:tngc in control wlut~on

339

Tetrahymena membrane potential Table 3. Temperalure and resting pokntial changes in Na+ solution R.P., (mV)

-21

- 15.5 -18 -30 -12

Temperature solution

R.P 1 (mVj -14 ~ 18.8 ~ 17.3 -19 ~ I9 - I4 ~ 27.5 23.5 --II -31 -21 -26.5 ~ 12.2 -16.0 -31.2 -17

und resting potential

R.P. bV1 - 16.5 - 17.9 - 17.7 -?I 25 -15 -15 -- 18.25 ~ 18.25 ~ 13.5 ~ 26.5 ~ 24.5 -23.75 ~ 13.8 - 17.0 - 33.6 - 14.5 I6 ~ 19.2 1.4

The e&t of Ouabain response to temperature

( C)

17.5 I6 I7 25.5 14 16.5 13 16.5 20 15.5 23 I5 20 20 I7 17.5

on the membrane change

Ql0

AC

13.75 20.5 I5 20.5 18.5 14.8 19.0 2 I .o I7 20.75 19.0 20 17.5 17.5 22 20.75 I6 18.6 0.6

IO 25 I? 15.5 23 I3 25 25.5 I4 26.0 I5 25 I5 I5 21 24

0.67 0.2 0.175 0.4 0.73 0.57 0.71 I.17 0.83 0.86 0.88 0.55 0.67 0.34 0.72 0.67 I6 0.63 0.07

1.51 1.12 I.11 I.21 I.65 I .47 I .4x l.Y4 1.89 I .39 I .44 1.26 1.64 I .22 1.24 1.47 I6 I .44 0.06

Control solution

changes in Nat

Twenty millimoles of NaCl was added to control solution in order to determine whether Na+ is involved in a metabolic contribution to the resting potential. Chloride ions are assumed to be passively distributed (Dunham and Child, 1961). The results of sixteen independent responses are summarised in Table 3 and an example is shown in Fig. 3(b). The mean resting potential of cells in this solution is lower than that established for 139 cells in control solution (P < 0.01). It is therefore probable that Na+ contributes an electrodiffusion potential to the total membrane potential. The Qlo value of membrane potential responses to temperature changes in Na+ solution was 1.44 + 0.06 (n = 16), a significantly greater response than in control solution (P < 0.001). The Qlo value is also greater than that predicted by electrodiffusion theory (P < 0.001). The frequency distribution histogram (Fig. 5a) illustrates the shift in the modal classes of potential responses from 0.1 to 0.4 mV/“C in control solution of 0.6-0.9 mV/“C in Na+ solution. The frequency distribution of Q,, values (Fig. 5b) shows a similar shift. This increase in potential responses and Q1,, values suggests that Na+ is involved in an electrogenic component of the resting potential of Tetrahymena.

A mV

t

T, ( C)

(a)

Na+sdution : 32L 2E I: _ 0

01

I 02

I 0304

I 05

06

07

08

09

Temperature dependent potential

I I

II

changes

1 12131415

I

I

( mVPC 1

Control solution

potential

The addition of Ouabain at 10m5 M resulted in a marked reduction of the membrane potential changes associated with temperature changes. To illustrate this a typical membrane potential change in Na+ solution (Fig. 4b) is compared with that in Na+ solution containing 10ms M Ouabain (Fig. 4~). Tables 4 and 5 summarise the results obtained in cells in control and Na+ solution containing lo-’ M Ouabain. In control solution with Ouabain, the Q,, value is 1.14 k 0.06 (n = 6) compared to 1.22 f 0.03 (n = 25) in control solution alone. This reduction is not significant at the 1% level (0.25 > P > 0.1). However the reduction by Ouabain ofthe Qlo value in Naf solution from 1.44 k 0.06 (n = 16) to 1.09 k 0.01 (n = 6) is

Na’solution

I1112131415161718192 O,,

values

Fig. 5. (a) Frequency chart showing the distribution of temperature induced resting potential changes (mV ‘C-‘) in control solution and with 20mM NaCl added. (b) Frequency chart showing distribution of Q,, values obtained in control solution and Na+ solution.

J. G.

C‘ONN0LL.Y

significant (P < 0.01). The reduction of Qto values by Ouabain suggests that a Na+-K+ ATPase may contribute electrogenically to the membrane potential in Tetrahymena. The increased significance of this reduction in the Na+ solution as compared to control solution further implicates Na+ as one of the ions involved in electrogenic transport. Despite the action of Ouabain the potential responses in Tables 4 and 5 were still significantly greater than the values predicted by electrodiffusion theory (control solution and Ouabain, P < 0.001, Na’ solution and Ouabain, P < 0.01). Thus Tetrahymena may also have electrogenic mechanisms which are not sensitive to Ouabain.

In Nature, free living freshwater ciiiates such as Tetrahymena occupy a wide variety of habitats which constantly present osmotic and ionic challenges to the internal environment of these cells. In carrying out the processes of feeding, digestion, pinocytosis and osmoregulation Tetrahymena must interact with an uncontrolled external medium, unlike the more protected excitable cells in mammals. Thus it may be expected that Tetrahymena should have powerful and rapidly responsive mechanisms for maintaining the ionic gradients across its membranes. This would also be expected of Paramecium, a closely related ciliate. The electrical properties of Paramecium (Naitoh and Eckert, 1968, 1972; Kung and Saimi, 1982) are very similar to those of Tetrahymena (Connolly and Kerkut, 1981, 1983). Paramecium has a resting potential of approximately - 30 mV in a solution of the same ionic composition as the control solution used in this present work ; while Table

5. Temperatureand

resting

potential

changes

and

ci.

A.

KkKII

I

Tetrahymena has a resting potential of 24.2 _t 0.8 (n = 139). The input resistance of ~u~~~~?t~~~~z~~~ ~~[~~~~~~{~~l was approximately 64 M (Naitoh and Eckert, 1968) which Machemer and De Peyer (1977) calculate to be equivalent to a specific membrane resistance of 7 x lo40 cm’. Using the same method of calculation, which is based on the Fortner (1925) formula for surface area, Tetrahymenu corax, with an input resistance of 47.8 f 2.9 MfS (n = 26), gives a specific membrane resistance of 3.2 x ItI4 Rem”. These resistances are a factor of 10 greater than more specialised cells such as Helix pomatiu (Meves, 1968). The similarity between Tetrahymena and Paramecium implies that the conclusions drawn from this present work on Tetrahymena may have a general application to Paramecium. The effects of Ouabain addition at lO_ ’ M were similar to those observed in other preparations such as snail neurons (Gorman and Marmor, 1970). The concentration used ensured that any reserve capacity (Akera et a[., 1981) of a Nat-K*-ATPase would be inhibited but that non-specific conductance increases associated with greater Ouabain concentrations (Garrahan and Rega, 1972) would be avoided. The fact that there was no significant reduction in membrane resistance after Ouabain addition supports the assumption that Ouabain is mainly acting in a specific manner. The average ‘t;, change in resting potential induced by 10e5 M Ouabain was 34.8 t. 4.6 (n = S), and at 5 x lo-’ Ouabain, the change was 27.1 i X.9”,, 0%= 3). An average of these results gives 3 I .9 f 4.6”,,. Thus as much as 309;, of the resting potential in Tetrahymena may be due an electrogenic Na ’ OK’ ATPase. Ouabain is a specific inhibitor of the Nat -K (Mg2’)-ATPase(Schatzm~~nn, 1953)and itsassociated in control

solution

containing

20 mM

Ouabain R.P,. hlVI

R.P.Z

R.P.

(mVl

(mV)

“- 20

-21.2

-20.6

14

-26

-25.2

-25.6

24

-14.0

-13.25

15

~ 24.5 _ 17.0

-25.0

22

-

21

-2x

-28.5

-25 -29

12.5 5 18.2

17.6

n ;r SE

A mV

6 -218 2.3

22

I*;a Cl and

IO

’

Tetrahymena membrane potential phosphatase activity (Inturrisi and Titus, 1970). However Andrus and Giese (1963) found that 10m3 M Ouabain did not affect the restoration of Na+ and K’ in Tetrahymena which had been exposed to cold, although this concentration of Ouabain might be expected to produce some non-specific effects. Conner et al. (1963) did find an Mg’+ dependent ATPase in Tetrahymena pyriformis whose activity could be enhanced by Naf and K+ by l&15%. However this enzyme was not sensitive to Ouabain. Baugh et al. (1976) found no evidence of the Na+-K+(Mg*+)ATPase in ciliary preparations from Tetrahymena, nor Doughty (1978) in Paramecium. Dentler (1977) detected a Nat-K’(Mg’+)-phosphatase in the ciliary basal bodies of Tetrahymena, but not an ATPase. These negative results are not conclusive however, since the most probable site for a Na+-K+(Mg”)ATPase, the contractile vacuole (CV), has not been investigated. A Na+-K+(Mg’+)-ATPase has been identified in Entamoeba invadens (McLaughlin and Meerovitch, 1975). Furthermore, Boggs and Wade (1972) in a study on the anterior vacuole of Paramecium multimicronucleatum, found that Ouabain slowed the activity and doubled the diameter of the CV. The nature of the mechanism producing these effects is uncertain, but it does suggest that a Nat-K’(Mg’+)-ATPase may be present in the CV region. The use of temperature changes to determine whether there is an active component in a membrane potential assumes parameters such as membrane permeability, resistance and internal ion concentrations remain constant over the temperature range investigated. The fact that there is no significant difference between results in Table 2 whose mean experimental temperature was above the average for the whole group and those below it suggests that if there are changes in cell properties these changes must be linear. Furthermore Ouabain specifically reduced membrane potential and Qr,, values. If the potential change induced by temperature was solely due to a change in a membrane constant Ouabain at the concentration used would not affect it. In Anisoderis (Marmor, 1971) and Aplysia (Russell and Brown, 1972) pNa+ : pK+ increases with warming. This would be a depolarising influence if the change occurred in Tetrahymena, yet an increase in the hyperpolarising response is observed upon warming in 20 mM Na+. Cooling Tetrahymena from 25 to 6°C reduces [K’li by 50% but increases [Na+li 2-fold (Andrus and Giese, 1963). Thus the effects of changes in internal ion concentration on membrane potential tend to cancel each other out. Overall, temperature dependent changes in electrophysiological properties would oppose metabolic rate changes in their effect on membrane potential. This is best illustrated in Anisodoris (Gorman and Marmor, 1970) and Aplysia (Carpenter, 1970; Marchiafava, 1970) where the application of Ouabain unmasks changes in membrane parameters and the hyperpolarising effect of warming is reversed to a depolarising effect. In conclusion, it can be said that non-metabolic temperature dependent changes contribute little or more probably detract from the potential changes observed in these experiments. Therefore monitoring the response of the membrane potential to temperature changes is a useful indication

341

of the importance of metabolism in the immediate maintenance of resting potential. The Qro values obtained for Tetrahymena by the above experimental approach in control (Qro = 1.22) and Na+ solutions (Qre = 1.44) were clearly and significantly greater than electrodiffusion theory alone would predict (Qie = 1.035). This is also the case in Paramecium (Yamaguchi, 1980) where a 10°C change in temperature gave a 2 mV change in potential (Qi,, = 1.07). However the potential measurements were made some time after the temperature changes so that adaptation may have reduced the potential change observed. These results imply that there is an electrogenic contribution to the membrane potential. The enhancement of the Qro values by Na+ addition from 1.22 to 1.44 suggests that Na+ is involved in electrogenic transport. The significant reduction of the Qlo value in Na+ solution (1.44 + 1.09) by Ouabain is strongly suggestive that a Na+-K+-ATPase is involved in the potential responses. The insignificant (at 1% level) reduction of Qlo values (1.22+ 1.14) by Ouabain in control solution probably reflects the reduced activity of the Na+-Kf-ATPase in the absence of Na+ substrate. Even in the presence of Ouabam, however, the Qlo values were still significantly greater than electrodiffusion theory allows. Other mechanisms which may be involved are a Na+Ca*+ exchange (Browning and Nelson, 1976; Connolly and Kerkut, 1983) or the various cotransport mechanisms in Tetrahymena which involve Na+ such as Na+ and sugars (Cirillo, 1962; Aomine, 1974). However the Na+-Ca*+ exchange may be largely inactivated at resting potential so that this remaining Qlo difference is probably the sum of many small-scale processes. The resting potentials of both Tetrahymena (Connolly and Kerkut, 1981) and Paramecium (Naitoh and Eckert, 1968) are strongly dependent upon external cations. The resting potential of Tetrahymena is depolarised by 28.3 mV for a lo-fold increase in [K’], compared to 30 mV in Paramecium. A lo-fold increase in [Ca’+], will lead to a 9.7 mV depolarisation in Tetrahymena and an 11 mV change in Paramecium. However these potential changes may not be the simple result of permeation of ions through the membrane. Protozoans have dilute intracellular fluid so that an influx of ions may lead to a change in tip potential which would contribute to the observed potential change (Satow, 1982). Eckert and Brehm (1979) discuss the possibility that increasing external cation concentrations results in surface charge neutralization and an increase in the potential difference across the membrane itself. When this is restored, presumably by conductance changes, the result is a depolarisation in the resting potential measured between the intracellular and extracellular bulk solutions (Kamada, 1934; Naitoh and Eckert, 1968; Connolly and Kerkut, 1981) an increase in input resistance and a positive shift in I-V relations (Satow and Kung, 1976; Eckert and Brehm, 1979; Satow, 1982). The rate of pumping of electrogenic transport mechanisms will also alter as the substrate concentration varies. Thus estimates of specific ion permeabilities based on electrophysiological data on bulk ion studies are liable to be inaccurate. Browning and Nelson (quoted in Machemer and DePeyer, 1977) have given an estimate

341

1. C;.

COI\;NOLLY

of pK

: pNa : pCa” ’ 0T I : 0. I : 0. I. However active transport of K ’ and Na’ probably exaggerates the pK: pNa ratio. so that the correct ratio of permeabihties may be the order of magnitude of 1 : 1 : 0. I. Compared to other excitable cells the ciliates are generally much less permeable. Tetrahymena has a specific resistance of 3 x IO4 Qcm’ whereas Aplysia neurone has a resistance of 2.2 x 1O”Q cm’ (Taut, 1955; Fessard and Taut, 1956). Stoner and Dunham ( 1970) have shown that ammo acids may be modified to maintain the osmotic balance between the internal and external environment. thus altering the Donnan equilibrium within the cell. Although [CL Ii is maintained at a lower level than [Cl I,, (Dunham and Child. 1961) CL is thought to contribute little to the resting potential in Paramecium (Naitoh and Eckert. 1974). Hence the major influences on resting potential would be [Na ‘I,, [K ‘I,, and the rate of pumping by electrogenic transport mechanisms. An equation describing the steady state resting potential of Tetrahymena will probably be of the form (based on Moreton, 1969) :

and

C.

A.

KLKKIIT

1970). This implies that these cells may screen electrtcal activity in localised areas by separating these systems from the buik solution by Il~ernbr~~lles of high r&atante. Such an arrangement has been proposed for the ciliary plaques (Connolly and Kerkut. 1983). which may screen Na+ : Ca’+ exchange. Biochemical investigations in both Tetrahymena (Nozaua and Thompson. 1971) and Paramecium (Kaneshiro ct ol.. 1979) have demonstrated that membranes from functionally and an~itomical~y different parts of the cell differ in contposition and therefore may dilfer in permeability. Furthermore in Paramecium voltage sensitive Ca2 ’ channels are known to bc located in the cilia (Opura and Takahashi. 1976: Dunlap. 1977) and there is assymmetric distribution of mechanoreceptive channels in both T~trahymena (Connolly and Kerkut, 1981) and Paramecium (Naitoh and Eckert. 1972). If specialised membranes do produce electrical heterogeneity and screening in Tetrahymena, then the same phenomena may occur in more advanced excitable cells such as mammalian pyramidal cells. The electrical

.-.--.-

where

and M is the ion flux across the membranes. This equation assumes that H +, OH and PO: make little contribution to the resting potential and that the permeabilities of Mg’ + and Ca’+ are very small compared to K ’ and Nat. From these studies it would seem that the electrogenic pump term, M ,RT/FV, may account for up to 30”,,, of the membrane potential. More work involving changes of [K * 1, in the presence and absence of Na+, Ouabain and cold is necessary to evaluate the pK ’ : pNa’ ratio accurately. However, as noted above, whatever technique is used to estimate ion permeabilities tends to affect the value obtained. Estimating ion permeabilities is further complicated by the physical structure of Tetrahymena. The somatic membrane is underlain by alveolar sacs which make it effectively a 3-layered structure (the pellicle). The sacs may also provide a buffered zone between the bulk of the intracellular tluid and the external solution. The alveoli do not extend into the intraciliary space, so that the permeability of the ciliary membrane may be greater than that of the pellicle. Furthermore the alveoli and basaI plate compartment the cilia from the cell to some extent and this could lead to a partial screening of electrical events in the cilia. Yamaguchi (1960) noted that potential oscillations with an amplitude of 18 mV were observed when the recording electrode was placed near the anterior contractile vacuole of Paramecium. Simiiar oscillations have been ~~ccasionally recorded in Tetrahymena (Connolly and Kerkut, 1981) and Zoothamnium (Moreton and Amos, 1979). The fact that such a large voltage change was not seen when recording distally to the CV suggests that the cell is not isopotential. However the inference drawn from measurements made with two recording electrodes placed in the cell soma was that the bulk of the somatic intracellular Ruid is isopotential (Eckert and Naitoh,

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and G. A. KI,KKI

1

den akti\en kaliumund N~ttrlumtranrport durch dtc t