Effect of Acetylcholine on Postjunctional Membrane Permeability in Eel Electroplaque N. L. L A S S I G N A L and A. R. M A R T I N From the Department of Physiology, University of Colorado Medical Center, Denver, Colorado 80262

ABSTRACT Acetylcholine (ACh) was applied iontophoretically to the innervated face of isolated eel electroplaques while the membrane potential was being recorded intracellularly. At the resting potential (about -85 mV) application of the drug produced depolarizations (ACh potentials) of 20 mV or more which became smaller when the membrane was depolarized and reversed in polarity at about zero membrane potential. The reversal potential shifted in the negative direction when external Na + was partially replaced by glucosamine. Increasing external K+ caused a shift of reversal potential in the positive direction. It was concluded that ACh increased the permeability of the postjunctional membrane to both ions. Replacement of C1- by propionate had no effect on the reversal potential. In Na+-free solution containing glucosamine the reversal potential was positive to the resting potential, suggesting that ACh increased the permeability to glucosamine. Addition of Ca ++ resulted in a still more positive reversal potential, indicating an increased permeability to Ca ++ as well. Analysis of the results indicated that the increases in permeability of the postjunctional membrane to K+, Na +, Ca ++, and glucosamine were in the ratios of approximately 1.0:0.9:0.7:0.2, respectively. With these permeability ratios, all of the observed shifts in reversal potential with changes in external ionic composition were predicted accurately by the constant field equation. INTRODUCTION

T h e major effect o f acetylcholine (ACh) at the end-plate o f vertebrate skeletal muscle is to p r o d u c e an increase in m e m b r a n e permeability to Na + a n d K + (Takeuchi and T a k e u c h i , 1960). At the resting m e m b r a n e potential the main consequence o f these permeability increases is an inward flux o f Na +, as the m e m b r a n e is at or near the equilibrium potential for K +. At m o r e depolarized m e m b r a n e potentials the inward m o v e m e n t o f Na + is a c c o m p a n i e d by o u t w a r d m o v e m e n t o f K +, the two ionic currents b e c o m i n g equal and opposite at the socalled reversal potential, where the net A C h - i n d u c e d c u r r e n t is zero. Estimates o f the reversal potential at the m o t o r end-plate r a n g e f r o m 0 to about - 1 5 mV (delCastillo a n d Katz, 1954; T a k e u c h i a n d Takeuchi, 1960; T a k e u c h i , 1963a, b; K o r d a L 1969; Gage a n d McBurney, 1972; Magleby a n d Stevens, 1972; Dionne and Stevens, 1975). A l t h o u g h the postjunctional m e m b r a n e o f the eel electroplaque is analogous to the m o t o r end-plate and has been used as a source o f THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 70,

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purified ACh receptors (Olsen et al., 1972; Klett et al., 1972; Patrick and Lindstrom, 1973), its electrophysiological properties are m u c h less well defined. In particular, the reversal potential for A C h - i n d u c e d m e m b r a n e currents has not been d e t e r m i n e d accurately, n o r have the permeability changes u n d e r l y i n g such currents. With bath-applied agonists the postjunctional c u r r e n t does not a p p e a r to reverse at any potential (Lester et al., 1975), a p p a r e n t l y because the overall c o n d u c t a n c e o f the drug-activated channels is r e d u c e d to zero by extreme m e m b r a n e depolarization. Because of this it has been necessary to estimate the reversal potential by extrapolation o f the relation between agonistinduced c u r r e n t a n d m e m b r a n e potential, obtained in the region o f m e m b r a n e potential where the channels are still open. Similarly, extrapolation has been used to estimate the reversal potential o f junctional potentials evoked by nerve stimulation (Ruiz-Manresa and G r u n d f e s t , 1971). In the experiments to be r e p o r t e d here, ACh was applied iontophoretically to the postjunctional m e m b r a n e o f the electroplaque (delCastillo et al., 1972) at various m e m b r a n e potentials. Using this technique, we were able to reverse the ACh potential at about zero m e m b r a n e potential. T h e reversal potential was altered by changes in Na +, K +, or Ca ++ concentrations in the bathing solution, but not by changes in extracellular CI-, suggesting that the permeability changes p r o d u c e d by A C h were similar to those at the m o t o r end-plate. Preliminary reports o f the present results have a p p e a r e d elsewhere (Lassignal and Martin, 1976a, b). MATERIALS

AND

METHODS

Single electroplaques were dissected by the method of Schoffeniels and Nachmansohn (1957) and mounted horizontally between two chambers (Fig. I). The innervated face was exposed to the upper chamber through a small (0.43 mm 2) hole, while the entire noninnervated face was exposed to the lower chamber. A thin layer of cured Sylgard resin (Dow Corning Corp., Midland, Mich.) was applied to the bottom of the upper chamber and uncured resin was used as a seal between this surface and the innervated face of the cell. All experiments were done at 22°C. Recording micropipettes were filled with 4 M K-acetate and had resistances between 15 and 40 Mfl. Tip potentials ranged from - 6 to -14 mV in the various experimental bathing solutions. The membrane potential of the innervated face was measured by two micropipettes glued together so that their tips were staggered by about 80/zm. The cell was impaled by the leading electrode tip while the other remained just outside; the difference in potential between the two tips was then taken as the membrane potential. The membrane potential was altered by passing current between the two chambers through calomel half-cells. Current was monitored by a current-to-voltage converter between the calomel electrodes in the upper chamber and ground. Either voltage clamp or current clamp techniques could be used, but only the latter was employed in the experiments to be reported here. As the resistance of the innervated membrane was quite low (approximately 8 f~ cm2), the potential across the fluid resistance between the two recording pipette tips could contribute significantly to the apparent change in membrane potential when current was passed through the cell. This was compensated for by adjusting the slider on the 5K potentiometer connected to point A (Fig. 1) so that no voltage was produced by applied currents when the leading electrode tip was just outside the cell (cf. Hodgkin et al., 1952).

LASSIGNALAND MARTIN AceOlcholine and Postjunctional Membrane Permeability

25

T h e normal saline solution had the following composition (mM): NaCI, 160; KCI, 2.5; CaCI2, 2.0; MgCI2, 2.0, buffered to p H 7.4 with the Na + phosphate (1.5 mM). In addition, all solutions contained 0.5 ~g/ml tetrodotoxin to block action potential initiation. Nadeficient solutions were made by replacing NaCI with glucosamine-HCl in equiosmolar quantities. T h e p H o f such solutions was adjusted by addition of N a O H . In preliminary experiments, replacement o f Na by Tris hydroxymethyi a m i n o m e t h a n e (Tris) or by methylamine was found to be unsuitable, as both a p p e a r e d to permeate the AChactivated channels. In experiments in which K + concentration was changed, sufficient glucosamine-HCl was substituted for NaCI in the control solution so that increases in KCI • -V m

5K R I VCIK 4,.

"

-

l-'vH~ CC

IOK

A

•

~7

Im

IC3

FIGURE 1. Experimental a r r a n g e m e n t . Single electroplaque was m o u n t e d between two chambers and m e m b r a n e potential measured by intracellular electrode (connected to amplifier AI) with reference to second microelectrode just outside the m e m b r a n e (A2). O u t p u t from IC2 used to pass c u r r e n t across the cell for depolarizing cell m e m b r a n e . C u r r e n t was measured by current-to-voltage converter (IC3). B, bucking potentials to set inputs o f [C1 to zero. R1 and R2 adjusted to provide unity gain at o u t p u t o f IC1 for signals from A1 and A2 with slider at point A. Moving slider away from point A compensated for voltage d r o p between microelectrode tips when current was passed t h r o u g h cell. All experiments reported here were with c u r r e n t clamp (switches in CC position). -Vm: m e m b r a n e potential output; Ira: m e m b r a n e c u r r e n t output. could be accompanied by equiosmolar decreases in glucosamine-HCl, thus keeping the Na concentration constant. CI concentration was reduced by substituting Na propionate for NaC1. After the cell had been impaled with the recording electrode, ACh was a p p l i e d iontophoretically from a micropipette filled with 4 M ACh (Nastuk, 1953), by using c u r r e n t pulses of 0.5-1.0 g,A and 50-60 ms duration. Although visual observation was obscured by the dense network o f blood vessels and nerves over the innervated face of the cell, it ~as usually possible to place the ACh pipette within about 20 t~m of the r e c o r d i n g electrodes. U n d e r these conditions variations in the responses to ACh application from one position to the next were more likely to be related to the relative success in penetrating the overlying tissue with the ACh pipette than to any intrinsic variations in sensitivity of the cell m e m b r a n e . When a suitable ACh potential was obtained it was superimposed on a succession o f

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membrane depolarizations of 600 ms duration (Fig. 2) to obtain the reversal potential (Er). In a given cell, Er for each solution tested was obtained by averaging the results of from one to five impalements. The solution was then removed and replaced after the chamber had been washed three times with the new solution. An equilibration time of 20 min was allowed in each solution before records were taken. The final Er for a particular solution was taken as the average of the results from all cells tested in that solution. RESULTS

Normal Solution In the n o r m a l saline solution the a v e r a g e resting potential o f the i n n e r v a t e d m e m b r a n e was - 8 8 . 2 --- 6.1 m V ( m e a n + SD, 18 cells). I o n t o p h o r e t i c application o f ACh p r o d u c e d a depolarization which rose to a peak in 75-200 ms. Such a r e s p o n s e is shown in Fig. 2 at a resting potential o f - 9 4 m V a n d at various levels of depolarization. As the m e m b r a n e was depolarized the response b e c a m e +33--%.~

'

'

-2----

-6a 200 mll

FIGURI~ 2. Responses to iontophoretically applied ACh at resting membrane potential (-94 mV) and various levels of membrane depolarization. Response reverses at about - 2 mV. Duration of ACh pulse indicated by artifact at beginning of response. smaller, d i s a p p e a r i n g at a b o u t zero m e m b r a n e potential a n d r e v e r s i n g with reversed m e m b r a n e polarization. T h e results f r o m the e x p e r i m e n t o f Fig. 2 are plotted in Fig. 3, which shows the relation between A C h potential a m p l i t u d e a n d m e m b r a n e potential. In this particular e x p e r i m e n t the A C h potential r e v e r s e d at a m e m b r a n e potential o f a b o u t - 3 mV. I n all e x p e r i m e n t s the relation b e c a m e m a r k e d l y n o n l i n e a r at m e m b r a n e potentials positive to the reversal potential. At positive m e m b r a n e potentials the a m p l i t u d e o f the r e v e r s e d A C h potential b e c a m e almost i n d e p e n d e n t o f m e m b r a n e potential a n d sometimes e v e n decreased r a t h e r than increased with increasing polarization b e y o n d +30 inV. This b e h a v i o r was not d u e to any rectifying p r o p e r t i e s o f the extrajunctional m e m b r a n e as a similar relation was o b s e r v e d w h e n A C h was applied u n d e r voltage c l a m p conditions: i.e. the j u n c t i o n a l c u r r e n t was not linearly related to m e m b r a n e potential (Lassignal a n d Martin, 1976a). In m a n y o f the c u r r e n t c l a m p e x p e r i m e n t s the a m p l i t u d e o f the r e v e r s e d A C h potential was small (1-4 mV) a n d it was necessary to obtain at least a 20-mV A C h potential at the resting m e m b r a n e potential in o r d e r to observe reversal. In the 18 e x p e r i m e n t s in n o r m a l solution the a v e r a g e reversal potential was + 1.9 - 5.6 m V .

LASSIGNALAND MARTIN Acetyleholineand Postjunctional Membrane Permeability

27

Effect o f N a + on Reversal Potential

In o r d e r to investigate the role o f Na + in the r e s p o n s e to A C h , extracellular Na + was replaced by g l u c o s a m i n e . T h i s ion was chosen because it is relatively i m p e r m e a n t at the f r o g m o t o r e n d - p l a t e (Maeno et al., 1977). I n addition, the effects o f changes in e x t e r n a l Na were tested in solutions in which divalent cations (Ca ++ a n d Mg ++) were eliminated, as Ca ++ is k n o w n to p e n e t r a t e ACh-activated channels at the m o t o r end-plate ( T a k e u c h i , 1963b) a n d the c o n t r i b u t i o n o f such Ca ++ c u r r e n t b e c o m e s relatively m o r e i m p o r t a n t as e x t e r n a l Na + is r e d u c e d . T h e control solution, t h e n , consisted o f 160 m M NaCI, 2.5 m M KCI, 1.5 m M Na + p h o s p h a t e b u f f e r ( p H 7.4). T h e low Na + test solution c o n t a i n e d 32 m M N a O H ,

A

55

!

3o 25

Es(mV) 20

15 I0

5

-,~o-,6~o-~o -~ -,io -~'o Em(mV)

"~5~

0

-I0

FIGURE 3. Amplitude of ACh response (Es) as a function of membrane potential (Era) in normal bathing solution. Arrow indicates resting membrane potential. Response reverses at about - 3 mV membrane potential. 128 m M glucosamine-HC1, 2.5 m M KCI, 1.5 m M Na + p h o s p h a t e b u f f e r ( p H 7.1). C h a n g e s f r o m control to test solutions and back were m a d e in b o t h c h a m b e r s . T h e results o f o n e such e x p e r i m e n t are p r e s e n t e d in Fig. 4. I n the control solution (filled circles) the reversal potential was +4 m V . R e d u c i n g extracellular Na + c o n c e n t r a t i o n by a factor o f 5 shifted the reversal potential in the negative direction to - 19 m V . I n a total o f 18 e x p e r i m e n t s the a v e r a g e reversal potential in the control solution was +4.7 -.+ 2.9 m V , a value not significantly d i f f e r e n t f r o m that o b t a i n e d f r o m the e x p e r i m e n t s in the solution containing Ca ++ a n d Mg ++, a n d the a v e r a g e resting potential - 8 8 . 7 - 5.0 m V . In the low N a + solution (24 cells) the a v e r a g e reversal potential was - 1 8 . 9 _ 4.7 m V a n d the a v e r a g e resting potential - 7 8 . 6 - 5.0 m V . T h u s a fivefold r e d u c t i o n in extracellular Na p r o d u c e d an a v e r a g e shift o f - 2 3 . 6 m V in reversal potential. Effect o f K + on Reversal Potential

T o test the possibility that K + c u r r e n t m i g h t c o n t r i b u t e to the ACh potential,

28

T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E 7 0 • 1 9 7 7

reversal potentials were m e a s u r e d in normal (2.5 mM) K + and 32 mM K ÷. Again, divalent cations were eliminated f r o m the control a n d test solutions and all solution changes were made in both the u p p e r and lower c o m p a r t m e n t s . T h e control solution contained 120 mM NaCI, 9.4 mM N a O H , 30 mM glucosamineHCI, 2.5 mM KC1, 1.5 mM Na ÷ p h o s p h a t e buffer (pH 7.1), and the test solution 130 mM NaCI, 32 mM KCI, and 1.5 mM Na ÷ p h o s p h a t e (pH 7.1). In 12 experiments the average reversal potential in the control solution was - 1.5 -+ 2.9 mV with an average resting potential o f - 8 3 . 7 -+ 3.9 mV. In 10 experiments in the high K + solution the average resting potential was r e d u c e d to - 3 1 . 9 +- 3.2 mV and the reversal potential increased to +3.0 +- 3.4, not significantly different

e"'X

~28 Es(rnV) 2..4 20 16

12

" ErnlmV)

FIGURE 4. Effect of reducing extracellular Na + on reversal potential. Closed circles: ACh response as a function of membrane potential in normal (161.5 mM) Na +. Reversal potential about +3 inV. Open circles: responses in reduced (33.5 mM) Na +. Reversal potential is shifted to about -19 inV. Na + was replaced by glucosamine. f r o m that in the control solution. F r o m this result it could not be d e t e r m i n e d whether or not K + flux contributed to the ACh potential. T h e lack o f a shift in reversal potential is not necessarily an indication that K + is not involved in the response. I f one assumes that the reversal potential is d e t e r m i n e d by the constant field equation (Goldman, 1943), then little shift in the reversal potential might be expected (Anwyl and U s h e r w o o d , 1975). This may be appreciated intuitively by considering that increasing external K + not only shifts the K + equilibrium potential to a more positive value but also increases K + c o n d u c t a n c e (assuming constant K + permeability). T h u s , when the m e m b r a n e is depolarized the two effects tend to cancel, a n d the o u t w a r d K + c u r r e n t (and hence the reversal potential) may be similar to that in the control solution. However, the G o l d m a n equation predicts that the effects o f changes in K + concentration

LASSlGNAL AND MARTIN

29

Acetylcholine and Postjunctional Membrane Permeability

should be m o r e a p p a r e n t in low Na + solutions. Consequently reversal potentials were m e a s u r e d at the same two K + concentrations in solutions c o n t a i n i n g 33.5 mM Na + r a t h e r than the n o r m a l 161.5 mM. T h e control solutions contained 32 mM N a O H , 2.5 mM KCI, 128 mM glucosamine-HC1, 1.5 mM Na + p h o s p h a t e buffer, and the test solution contained 32 mM N a O H , 32 mM KCI, 100 mM glucosamine-HCl, 1.5 mM Na + p h o s p h a t e . Both solutions were at p H 7.1. Results obtained in one e x p e r i m e n t are shown in Fig. 5. In the solution containing n o r m a l K + the average resting poterttial was - 7 8 . 6 +- 5.0 mV and the reversal potential - 18.9 -+ 4.7 m V (24 cells). I n the high K + solution (18 cells) the average m e m b r a n e potential was r e d u c e d to - 2 9 . 3 - 2.4 m V and the average reversal potential shifted in the positive direction to - 1 0 . 3 - 2.6 mV.

Es(mV) 5 0 0

4

0

3 2 I C

-I()0 .

.

.

.

.

.

.

.

I

I0

EmlmVl FIGURE 5. Effect of K+ on reversal potential. Open circles: amplitudes of responses in low-Na, glucosamine solution with normal (2.5 mM) K +. Closed circles: responses in high (32 raM) K+. Reversal potential is shifted from about -23 mV to - 1 0 mV by increasing extracellular K+. Effect of Cl- on Reversal Potential T h e possibility that CI- might p e r m e a t e the ACh-activated channels was tested by c o m p a r i n g responses in the control solution (160 mM NaCI, 2.5 mM KCI, 1.5 mM Na + phosphate) with those in a test solution in which the NaCI was replaced by Na + p r o p i o n a t e . In f o u r e x p e r i m e n t s in which the test solution was present in the u p p e r c h a m b e r the average resting potential was - 8 0 . 6 - 1.5 m V and the reversal potential +5.0 _ 3.4 inV. T h e reversal potential was not significantly different f r o m that obtained in the control solution (+4.7 - 2.9 mV, see section on Na+), indicating that CI- did not contribute to the A C h response. Glucosamine and Ca ++ In o r d e r to analyze the results obtained with Na + and K + it was necessary to d e t e r m i n e to what extent, if any, glucosamine contributed to the A C h response. T o investigate this point the cell was bathed on both sides with a solution

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containing 140 m M g l u c o s a m i n e - H C l , 32 m M K O H , 1.5 m M K ÷ p h o s p h a t e b u f f e r ( p H 7.1). T h e results o f one such e x p e r i m e n t are plotted in Fig. 6 ( o p e n circles). At the resting potential o f - 3 0 m V a small A C h potential was obtained which b e c a m e r e d u c e d in a m p l i t u d e on depolarization a n d r e v e r s e d at a m e m b r a n e potential o f a b o u t - 2 1 m V . I n 13 such e x p e r i m e n t s the a v e r a g e resting potential was - 28. 3 -- 5.0 m V a n d the average reversal potential - 2 2 . 2 +- 5.1 m V . T h u s the reversal potential was a b o u t 6 m V positive to the resting potential. I f we assume that the resting m e m b r a n e potential was at or n e a r the K + equilibrium potential, t h e n the only ion p r e s e n t which would be e x p e c t e d to

Es(mV)

3.0 2.5! 2.0

0.5 I

,°.v, . Lo O

-I .5 -2.0

FXGURE 6. Open circles: amplitudes of responses in Na+-free glucosamine solution containing 32 mM K +. Reversal potential (-22 mV) is positive to resting membrane potential (-30 mV), suggesting that ACh increases membrane permeability to glucosamine. Closed circles: 20 mM CaCI2 added to bathing solution shifts reversal potential to about - 1 4 mV, indicating increased permeability to Ca ++ as well. carry any significant a m o u n t o f inward c u r r e n t was glucosamine. It was concluded that the ACh-activated channels were slightly p e r m e a b l e to this ion. In 5 o f the 13 e x p e r i m e n t s p a r t o f the glucosamine in the Na+-free solution was replaced by Ca ++ to d e t e r m i n e the effect o f the latter on the reversal potential. T h e test solution c o n t a i n e d 20 m M CaCI=, 105 m M g l u c o s a m i n e - H C l , 32 mM K O H , b u f f e r e d to p H 7.1 with 2.0 mM N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' ethanesulfonic acid (HEPES). Fig. 6 illustrates the effect o f a d d i n g the test solution to the u p p e r c h a m b e r in o n e e x p e r i m e n t . T h e resting potential ( - 2 9 mV) was essentially u n c h a n g e d ; the reversal potential (closed circles) shifted in the positive direction by a b o u t 7 m V to - 1 4 m V . I n the five e x p e r i m e n t s the a v e r a g e resting potential was - 3 4 . 5 - 3.4 m V a n d the a v e r a g e reversal potential - 1 2 , 9 -4- 3.3 m V . Addition o f 20 m M Ca, then, shifted the reversal potential by an a v e r a g e o f a b o u t +9 m V .

LASSIGNAL AND MARTIN

Acetylcholineand PostjunctionalMembranePermeability

31

ANALYSIS T h e results indicate that the effect o f ACh on the postjunctional m e m b r a n e was to increase its permeability to Na +, K +, and Ca ++, and, to a lesser extent, glucosamine. In the following analysis it will be assumed that the reversal potential Er is d e t e r m i n e d by the relative permeabilities o f the ACh-activated channels to the ions present according to the constant field equation ( G o l d m a n , 1943). A n o t h e r possible t r e a t m e n t o f the results will be c o n s i d e r e d subsequently. In Ca++-free solution, then, RT Ko + aGAo + bNao In (1) F KI + aGAt + bNat ' where R, T, and F have their usual meanings, K0, Ki, etc. r e p r e s e n t the concentrations (or m o r e strictly, the activities) o f the various ions outside and inside the cell, respectively. It should be n o t e d that glucosamine is not completely ionized at neutral pH; GAo, then, represents the ionic concentration, calculated by assuming that the ionized fraction exists as a m o n o v a l e n t cation and considering the solution to be electrically neutral. T h e constants a and b r e p r e s e n t Apaa/Ap~ and A/~a/A/~, respectively; i.e. the ratios o f the increases in permeability prod u c e d by ACh. In using Eq. (1) it was assumed that aGat was small with respect to the o t h e r terms in the d e n o m i n a t o r and this t e r m was ignored in the subsequent calculations. Er =

Internal Sodium and Potassium T h e main p r o b l e m e n c o u n t e r e d in applying Eq. (1) to the results is that accurate values o f Nal and Kl are not available. For the purposes o f the following analysis it was assumed that Nai was 15 raM, which would m e a n that in n o r m a l bathing solution the Na + equilibrium potential was +60 mV (see Discussion; Lassignal and Martin, 1976a). This value will be used in all o f the calculations p r e s e n t e d here. Similarly, all subsequent calculations (except those involving glucosamine and Ca ++ permeabilities) w e r e d o n e by using an estimated value for K~. This value was calculated by assuming that in high K + solutions the resting m e m b r a n e potential was equal to the K + equilibrium potential. T h e average resting potential in high (32 mM) K + was about - 3 0 mV, equivalent to an internal K + concentration o f 105 raM. In n o r m a l (2.5 raM) K +, this internal concentration results in a calculated K + equilibrium potential o f - 9 5 inV. T h e overall average resting potential in n o r m a l K + was - 8 4 . 5 inV. I f it is assumed that the resting potential is d e t e r m i n e d by the concentrations o f Na + and K + on either side o f the m e m b r a n e and the relative permeability o f the resting m e m b r a n e to the two ions according to the constant field equation, then the average resting potential is consistent with a resting Na + to K + permeability ratio o f 0.008, a value which is not unreasonable (Karlin, 1967). Permeability to Glucosamine Experiments like that shown in Fig. 6 (open circles) were used to d e t e r m i n e the constant a. In using Eq. (1) the assumptions already m e n t i o n e d were made, namely aGAl negligible, Nai = 15 raM, and Kt = K0 exp ( - E m F ] R T ) , whereEm is the resting m e m b r a n e potential in the high K + solution. In the e x p e r i m e n t s u n d e r consideration Na0 was zero. Eq. (1) then becomes

32

THE

R_T Er = - - I n

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Ko + aGAo K0e_em~.mT+ b(15)

where the ionic concentrations are in m M . O n r e a r r a n g i n g we obtain g o ( e (£r-Em)F/RT - a

1) + b(15)e e#'mT

=

GAo

A s s u m i n g b = 0.9 (see section on Na + below) and using a v e r a g e values forEm a n d Er f r o m 13 e x p e r i m e n t s gave a value o f 0.21 for a. Effect o f External N a + on Reversal Potential

T h e average reversal potential in the control solution was +4.7 inV. With the assumptions outlined above, the reversal potential should be given by Er

RT

7-

In

K0 + (0.21)GA0 + bNa0 105 + b(15)

(2)

It follows that 105 e ErFIRT K0 - (0.21)GA0 Nao - 15 e ErFIRT -

b =

-

(3)

I n the control solution the ratio o f Na + to K + permeability changes calculated f r o m Eq. (3) was 0.86. By using this value for b in Eq. (2), it was p r e d i c t e d that lowering the external Na + concentration f r o m 161.5 m M to 33.5 m M would shift the reversal potential in the negative direction by a b o u t 26 mV; i.e. to - 2 1 . 0 inV. T h e reversal potential observed e x p e r i m e n t a l l y in the low-Na + solution was - 1 8 . 9 -+ 4.7 m V , in good a g r e e m e n t with the prediction. Effect of External K + on Reversal Potential

W h e n reversal potentials were c o m p a r e d in n o r m a l (2.5 raM) a n d high (32 mM) K +, in the p r e s e n c e o f 131 m M Na +, little difference was observed. Such a lack o f effect o f changes in external K + on reversal potential is p r e d i c t e d by Eq. (2). In the control solution Er was - 1.5 m V , resulting in a calculated value for b o f 0.79. W h e n one used this value, the predicted reversal potential in high K + was 3.8 inV. T h e o b s e r v e d reversal potential was +3.0 --- 3.4 m V , not significantly d i f f e r e n t f r o m the predicted. S o m e w h a t larger changes in reversal potential with changes in external K + are predicted if the external Na + concentration is r e d u c e d . In the low (33 raM) Na + solution the reversal potential in n o r m a l K + was - 1 8 . 9 m V , giving a value of 1.0 for b. T h i s gave a predicted Er in high K + o f - 10.2 m V , in close a g r e e m e n t with the observed value o f - 1 0 . 3 - 2.6 m V . Permeability to Ca ++

In the e x p e r i m e n t illustrated in Fig. 6 it was f o u n d that a d d i n g 20 m M CaCI2 to the external solution resulted in a shift o f the reversal potential in the positive direction. I f we assume, at least for the m o m e n t , that the shift reflected a contribution o f Ca ++ c u r r e n t to the ACh response r a t h e r t h a n s o m e indirect effect of Ca ++ on a, t h e n we can calculate a value for the increase in Ca ++

LA$SIGNAL AND MARTtN

Acetylcholine and Postjunctional Membrane Permeability

33

permeability p r o d u c e d by ACh as a fraction o f the increase in K + permeability; i.e., we can calculate c = Apea/APK. At the reversal potential the sum o f all the ionic currents d u e to ACh application is zero or, in o t h e r words, IK + I N a + IGA + I c a

=

0.

(4)

I f we use the assumptions i n h e r e n t in the derivation o f the constant field equation (Goldman, 1943; H o d g k i n et al., 1952), then for any ion X with valence z, change in permeability &Px and internal and external concentrations Xl and X0, respectively, the ionic c u r r e n t due to ACh application at the reversal potential is given by Apxz2F2E r X|eZFErRT -- Xo Ix -

RT

e z~E,RT -

1

(5)

This expression can be substituted in Eq. (4) for each o f the ionic currents. In the experiments Na0 was zero, and the same assumptions were m a d e as previously, namely, Nal = 15 mM, G A i , and Cat negligible and Kl = Ko exp ( - E m F / R T ) . Making these substitutions and r e a r r a n g i n g , we arrive at an expression for c: C --

Ape a ApK

--

e E,FIRT + 1 [Ko(eC~,_Em~Fm T ~ 1) -- aGAo + b(15)eE'F/RT] .

4CAo

Values o f 0.21 and 0.9 were used for a and b, respectively. In the five experiments the a v e r a g e v a l u e s for Em and Er were - 3 4 . 5 and - 1 2 . 9 mV and c was calculated to be 0.70. DISCUSSION

All o f the shifts in reversal potential with changes in extracellular Na ÷, K ÷, and glucosamine a g r e e d well with those predicted by the constant field equation. Given that use o f the equation is then a p p r o p r i a t e , a value for b can be calculated for each o f the e x p e r i m e n t a l conditions. T h e average o f such calculations for the f o u r basic extracellular solutions (normal and low Na ÷ combined with n o r m a l and high K +) was 0.88 -_- 0.11, assuming a = 0.21. T h e overall results can be s u m m a r i z e d then, by saying that in the presence o f ACh the postjunctional m e m b r a n e behaves in a m a n n e r consistent with the constant field equation with increases in permeability to K +, Na ÷, Ca ++, and glucosamine being in the ratios o f 1.0:0.9:0.7:0.2, respectively. T h e good a g r e e m e n t between the shifts in reversal potential observed experimentally and those predicted theoretically d e p e n d s on the values used for internal Na + and K ÷ concentrations. Both values (Nai = 15 mM; Kl = 105 mM) are consistent with previous estimates for electroplaques. H i g m a n et al. (1964) f o u n d that the m e m b r a n e potential o f the innervated face followed the Nernst relation for K + with i n c r e a s i n g ' e x t e r n a l concentrations above 20 mM, so the assumption m a d e h e r e that the m e m b r a n e potential was n e a r the K + equilibrium potential in 32 mM K + seems reasonable. Previous estimates o f internal Na + have r a n g e d f r o m 10 mM to 30 mM (Schoffeniels, 1959; Karlin, 1967; but see RuizManresa and G r u n d f e s t , 1971), and/~a/PK for the resting m e m b r a n e has been estimated at about 0.01 (Karlin, 1967), in a g r e e m e n t with the present estimate o f 0.008.

34

T H E J O U R N A L OF GENERAL P H Y S I O L O G Y • V O L U M E 7 0 " 1 9 7 7

T h e results r e p o r t e d here differ in one i m p o r t a n t respect f r o m those r e p o r t e d previously with a variety o f muscle preparations (e.g., T a k e u c h i , 1963a, b; Ritchie and F a m b r o u g h , 1975). In general, shifts in reversal potential with changes in external Na + have not been consistent with those expected f r o m the constant field equation. Instead such shifts have been in a g r e e m e n t with those predicted by the conductance equation p r o p o s e d by T a k e u c h i and T a k e u c h i (1960) derived f r o m an electrical model o f the postjunctional m e m b r a n e . T h e equation states that the reversal potential should be given' by Er -

EK + rEsa l+r

,

(6)

where EK and ENa are the K+ and Na+ equilibrium potentials and r = A G N a / A G K , the ratio of the changes in conductance produced by ACh. It follows that

r =

Er EN~

E K - -

Er "

(7)

T h e analytical p r o c e d u r e is to d e t e r m i n e the conductance ratio f r o m Eq. (7) and then use this value in Eq. (6) to predict shifts in several potential with changes in external Na + a n d K +. With changes in external K +, it has been necessary to allow for the fact that K + conductance increases (and r decreases) with increasing concentration, a t r e a t m e n t equivalent to using the constant field equation. In contrast, shifts in reversal potential with changes in external Na + have been predicted accurately by Eq. (6), with a constant value for r, implying that Na + conductance is i n d e p e n d e n t o f concentration. This, in t u r n , suggests that Na + permeability increases as the external concentration is decreased. Various explanations for this a p p a r e n t anomaly have been p r o p o s e d (for summaries see: Ritchie and F a m b r o u g h , 1975; Gage, 1976). T h e problem does not arise in the present e x p e r i m e n t s , where the results obtained by changing external Na + were consistent with the constant field equation and not with the conductance equation. I f we apply the conductance equation to the present results, then in the control (Ca++-free) solution, assuming ENa = +60 mV and EK = --95 mV, a reversal potential o f zero results in a value o f r o f 1.58 (Eq. [7]). Using this value in Eq. (6) predicts a shift o f - 2 4 . 6 mV inE~ when going f r o m the control solution to the low-Na + solution, in a p p a r e n t a g r e e m e n t with the observed shift o f - 2 5 . 2 mV. However, in using Eq. (6), no account is taken o f the contribution o f the glucosamine in the low-Na + solution to the inward c u r r e n t associated with the response. This cannot be included in the conductance equation as the equilibrium potential for glucosamine is u n k n o w n , but f r o m Eq. (5) the glucosamine c u r r e n t would be expected to be approximately 87% o f the Na + c u r r e n t , in which case the conductance equation would predict a shift in E~ of only 9.1 mV when changing f r o m n o r m a l to low Na +. In s u m m a r y , then, the present results imply that Na + permeability in the ACh-activated channels is i n d e p e n d e n t o f Na + concentration, while previous experiments on muscle preparations suggest that Na + permeability increases when external concentration is r e d u c e d . T h e difference seems most likely to be

LASSIGNAL AND MARTIN Acetylcholine and Postjunctional Membrane Permeability

35

related to the fact that glucosamine was used h e r e as an N a + substitute while in previous e x p e r i m e n t s Na + c o n c e n t r a t i o n in the b a t h i n g solution was r e d u c e d by substituting sucrose for NaC1, thus r e d u c i n g the ionic s t r e n g t h o f the solution. This m i g h t alter the permeability characteristics o f the postjunctional m e m brane, for e x a m p l e by u n m a s k i n g fixed surface charges, in such a way as to increase Na + permeability (relative to that o f K +) a n d t h e r e b y m a k e Na ÷ conductance a p p e a r i n d e p e n d e n t o f concentration. In the p r e s e n t e x p e r i m e n t s the ratio o f Ca ++ to K + permeabilities was a b o u t 0.7; the Ca ++ to Na + permeability ratio was t h e r e f o r e a b o u t 0.77. I n e x p e r i m e n t s on the m o t o r e n d - p l a t e ( T a k e u c h i , 1963 b) the Ca ++ to Na ÷ c o n d u c t a n c e ratio was f o u n d to be about 0.08. If, in Eq. (5), we replace Er by Em and Ix by A G x ( E x - Era), w h e r e AGx is the increase in c o n d u c t a n c e for ion X a n d Ex is its equilibrium potential, t h e n we have an e x p r e s s i o n for the relation b e t w e e n permeability a n d c o n d u c t a n c e for ion X at the resting m e m b r a n e potential. Using this r~lation for Ca ++ a n d Na + a n d the s a m e a s s u m p t i o n s a b o u t Ca ++ distribution as those m a d e for the m o t o r e n d - p l a t e (Ca0 =- 2 raM; Cat = 1.18 raM; Eta = 6.7 mV), we arrive at AGca/AGNa = 0.06, a value similar to that r e p o r t e d previously. With a s o m e w h a t m o r e realistic a s s u m p t i o n that Cai = 1 /.tM (Eta = 100 mV), the c o n d u c t a n c e ratio is r e d u c e d to 0.03. Finally, the results r e p o r t e d h e r e d i f f e r f r o m those o b t a i n e d previously with b a t h application o f A C h agonists to the electroplaque (e.g., Lester et al., 1975) in that reversal o f the A C h r e s p o n s e was readily obtained. Difficulty in o b t a i n i n g reversal potentials with b a t h applications seems likely to be d u e not only to inaccuracies associated with the small m a g n i t u d e s o f the permeability c h a n g e s at e x t r e m e m e m b r a n e depolarization but also to changes in internal ionic concentrations d u r i n g the period o f application o f the agonist (Karlin, 1967). T h i s work was s u p p o r t e d by research g r a n t no. NS 09660 f r o m the National Institutes o f H e a l t h , B e t h e s d a , Md.

Received for publication 21 March 1977. REFERENCES

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