Sensitivity of the Sodium and Potassium Channels of Myxicola Giant Axons to Changes in External pH C. L. S C H A U F
a n d F. A. D A V I S
From the Departments of Neurological Sciences and Physiology, Rush Medical College, Chicago, Illinois 60612
M y x i c o l a giant axons were studied using standard voltage-clamp techniques in solutions whose pH values ranged from 3.9 to 10.2. Buffer concentrations of 50 mM or greater were necessary to demonstrate the full effect of pH. In acidic solutions the axon underwent a variable depolarization, and both the sodium and potassium conductances were reversibly depressed with approximate pKa's of 4.8 and 4.4, respectively. The voltage dependence of G~a was only slightly altered by acidic conditions, whereas there occurred large shifts in GK along the voltage axis consistent with a substantial decrease in net negative surface charge in the vicinity of the K + channels. The sodium and potassium activation rate constants were decreased by acidic conditions, but the results could not be described as a simple translation along the voltage axis. A B S ' r R A C "r
INTRODUCTION Alteration o f the e x t e r n a l p H basically has two distinct effects on m e m b r a n e p a r a m e t e r s . Various r e p o r t s (Hille, 1968, 1973; D r o u i n a n d T h e , 1969; Mozhayeva a n d N a u m o v , 1970, 1972; E h r e n s t e i n a n d F i s h m a n , 1971; Stillman et al., 1971; W o o d h u l l , 1973; D r o u i n a n d N e u m c k e , 1974; S h r a g e r , 1974) have shown that u n d e r acidic conditions b o t h the m a x i m u m s o d i u m a n d p o t a s s i u m c o n d u c t ances are reversibly d e p r e s s e d in a m a n n e r m o r e or less consistent with the titration o f a weak acid. In f r o g n e r v e t h e r e is general a g r e e m e n t that the pKa f o r the s o d i u m c o n d u c t a n c e is a r o u n d 5.2, whereas for p o t a s s i u m the pKa is n e a r 4.5. In squid the pK~ f o r the s o d i u m c h a n n e l was f o u n d to be 6.5, while GK was nearly u n a f f e c t e d by p H values a b o v e 5 (Stillman et al., 1971). H o w e v e r , in crayfish S h r a g e r (1974) f o u n d a pKa o f 6.3 for the p o t a s s i u m c o n d u c t a n c e , a n d c o m m e n t e d that the s o d i u m c o n d u c t a n c e was relatively less sensitive to c h a n g e s in p H . In addition to this effect on c h a n n e l c o n d u c t a n c e , a r e d u c t i o n in e x t e r n a l p H also has a m a r k e d effect o n the voltage d e p e n d e n c e o f a variety o f m e m b r a n e p a r a m e t e r s , a n d h e r e also the results d e p e n d on the p r e p a r a t i o n e m p l o y e d . I n f r o g n e r v e b o t h the s o d i u m a n d p o t a s s i u m c o n d u c t a n c e - v o l t a g e relations are shifted in the d e p o l a r i z e d direction as the solution b e c o m e s increasingly acidic, a l t h o u g h the effect m a y be s o m e w h a t g r e a t e r for the p o t a s s i u m c h a n n e l (Hille, THE JOURNALOF GENERALPHYSIOLOGY VOLUME67, 1976 ' pages 185-195 "
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1968; D r o u i n a n d T h e , 1969). T h e r a t e c o n s t a n t s g e n e r a l l y a p p e a r to b e s h i f t e d by a m o u n t s c o m p a r a b l e to t h e c o n d u c t a n c e s . I n c r a y f i s h a x o n s , h o w e v e r , t h e e f f e c t o f p H o n t h e r a t e c o n s t a n t f o r p o t a s s i u m a c t i v a t i o n is m u c h l a r g e r t h a n t h e effect on the conductance, and combined with a relative insensitivity of the s o d i u m c h a n n e l to p H , t h i s r e s u l t s in a n e a r l y c o m p l e t e t e m p o r a l s e p a r a t i o n o f t h e s o d i u m a n d p o t a s s i u m c u r r e n t s ( S h r a g e r , 1974). D e t a i l e d d a t a h a v e n o t b e e n reported for squid axons. B e c a u s e o f t h e s e v a r i a t i o n s in r e s p o n s i v e n e s s to p H f r o m o n e s y s t e m t o a n o t h e r , a n d t h e e x i s t e n c e o f c o m p l e t e d a t a f o r b o t h c h a n n e l s o n l y in n o d e , we felt it a p p r o p r i a t e to c a r r y o u t s i m i l a r s t u d i e s in v o l t a g e - c l a m p e d Myxicola g i a n t a x o n s . I n a d d i t i o n , s u c h s t u d i e s t e n d to c o m p l e m e n t t h e p r e v i o u s i n v e s t i g a t i o n o f m e m b r a n e s u r f a c e c h a r g e in this p r e p a r a t i o n ( S c h a u f , 1975). METHODS
Myxicola giant
axons were voltage clamped and the resulting data analyzed by methods described in detail elsewhere (Binstock and G o l d m a n , 1969; G o l d m a n and Schauf, 1972, 1973; Schauf, 1973, 1975). Compensated feedback was used in all experiments. T h e reference artificial seawater (ASW) solution had the composition: 430 mM NaCI, 10 mM KCi, 10 mM CaCi2, 50 mM MgCi2. In a few cases Mg ++ free seawater having various Ca ++ concentrations was used. T e m p e r a t u r e was maintained at 5.0 - 0.5°C. In the initial experiments 5 mM Tris maleate had been used as the buffer system. At this b u f f e r i n g capacity no changes in m e m b r a n e parameters could be observed after changes in solution p H . A nearly comparable insensitivity was f o u n d at 10 mM buffer. Only when the concentration o f Tris maleate (or Tris acetate in experiments at the most acidic p H values) was raised to 50 mM was the effect o f p H readily a p p a r e n t . A f u r t h e r increase in buffer concentration to 100 mM proved no m o r e effective than 50 mM. T h u s all the data r e p o r t e d here were obtained using 50 mM Tris maleate or Tris acetate with the p H adjusted by addition of HCI or N a O H just before an e x p e r i m e n t , and r e m e a s u r e d immediately after its use on an axon. T h e high t e m p e r a t u r e coefficient of Tris was taken into account. It should be pointed out that there was no detectable effect on m e m b r a n e parameters when the b u f f e r concentration was increased from 5 to 50 mM at the s t a n d a r d p H o f 7.8, only when buffer concentration was increased u n d e r acidic conditions. In the majority o f cases tetrodotoxin (TTX) was not routinely used to accurately obtain n o n s o d i u m currents. Rather peak transient and steady-state currents were corrected for the leak conductance measured d u r i n g a 100-mV hyperpolarization. However, in those experiments involving measurements o f the rate o f potassium activation, or steady-state sodium inactivation, T T X (10 -6 M) was used. In addition, a few experiments were done with T T X to ensure that there was in fact no significant difference in the conductance shifts measured using either method. A solution of ASW at a p H o f 7.8 -+ 0.05 served as the control for all measurements. T h e effects o f p H were usually complete within a few minutes, but we normally allowed the system to equilibrate for 15 min before r e c o r d i n g data. Each e x p e r i m e n t at an acidic or basic p H was bracketed by runs at the reference p H of 7.8. T h e m a x i m u m sodium a n d potassium conductances, as well as the half-maximal potentials (Vi sa and Vt K) were averaged for the bracketing runs at 7.8, and the relative conductance or relative voltage shift calculated using these averaged values. Axons in which there were very large differences between the bracketing values were assumed to have d e t e r i o r a t e d d u r i n g the course of the e x p e r i m e n t a n d the data were not used.
C. L. SCHAUFAND [7. A. DAVXS pHEffects on MyxicolaAxons
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RESULTS
Fig. 1 illustrates the effect o f an acidic solution on the m e m b r a n e c u r r e n t s in
Myxicola. R e d u c i n g the p H f r o m 7.8 to 4.6 causes an inhibition o f b o t h the p e a k transient a n d steady-state c u r r e n t s , t h o u g h the f o r m e r is slightly m o r e sensitive. T h e r e is a large decrease in the rate o f activation o f the p o t a s s i u m c u r r e n t which is b e t t e r s h o w n in Fig. 2 w h e r e 10 -e M T T X has b e e n used to eliminate m e m b r a n e s o d i u m c u r r e n t . A l t h o u g h it m a y be difficult to discern f r o m the r e c o r d s in Fig. 1, the time to p e a k i n w a r d c u r r e n t is significantly increased as well. H o w e v e r , in contrast to the results o f S h r a g e r (1974) o n crayfish axons, the s o d i u m a n d p o t a s s i u m c u r r e n t s do not b e c o m e t e m p o r a l l y s e p a r a t e d u n d e r acidic conditions. M e m b r a n e c u r r e n t - v o l t a g e relations at n o r m a l a n d acid p H are shown in Fig. 3, a f t e r correction f o r a linear leak. T h e s o d i u m l ( V ) c u r v e is d e c r e a s e d with little sign o f a voltage shift a n d with n o significant c h a n g e in the s o d i u m e q u i l i b r i u m potential (ENa). In contrast, the p o t a s s i u m I(V) shows a m a r k e d translation in the d e p o l a r i z e d direction a l o n g the voltage axis. In b o t h cases reversibility is excellent. It should be n o t e d that in Myxicola a x o n s a r e d u c t i o n o f the solution p H below 6.0 causes an a p p r e c i a b l e m e m b r a n e d e p o l a r i z a t i o n (range 4 - 1 2 mV), which is h o w e v e r c o m p l e t e l y reversible. T h e data show a large a m o u n t o f scatter, howe v e r , a n d it is impossible to d e m o n s t r a t e a m o n o t o n i c p H d e p e n d e n c e o f m e m b r a n e potential.
FIGURE 1. Voltage-clamp currents recorded in ASW at a pH 7.8 (top) and 4.6 (bottom) in response to depolarizations of 40, 50, 60, 70, 80, 90, and 100 mV from a holding potential of - 6 0 mV. Current and time scales are 0.3 mA/cm 2 and 2.0 ms, respectively. Temperature 5°C.
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FIGURE 2. Voltage-clamp current recorded in ASW + 10-8 M T T X at a pH 7.8 (top) and 4.6 (bottom) in response to depolarizations of 20, 40, 60, 80,100,120, and 140 mV from a holding potential of -65 mV. Current and time scales are 0.3 mA/cm2 and 2.0 ms, respectively. Temperature 5°C.
Effects of pH on Channel Conductance I n Fig. 4 we have plotted the m a x i m u m s o d i u m a n d p o t a s s i u m c o n d u c t a n c e s as a function o f solution p H , n o r m a l i z e d to the c o n d u c t a n c e s o b s e r v e d at the r e f e r ence p H o f 7.8. Data are only shown over the r a n g e 3.9-7.8, but were o b t a i n e d u p to a p H o f 10.2. W h e n we a v e r a g e d the values o f relative c o n d u c t a n c e at p H values above 7.8 we o b t a i n e d 1.01 -+ 0.03 for potassium (-+ SEM) a n d 1.03 - 0.04 for s o d i u m with no evidence for a n y systematic d e p e n d e n c e on p H (six experiments). T h e solid lines are calculated f r o m the dissociation o f a weak acid with a pKa o f 4.8 for G N a a n d 4.4 for GK. A l t h o u g h the solid lines m o r e or less describe the p H d e p e n d e n c e o f the m a x i m u m channel c o n d u c t a n c e , t h e r e seems to be s o m e systematic deviation. Points at p H values l a r g e r t h a n the pKa are below the line, while those at a p H m o r e acidic t h a n the pK~ are above. T h i s deviation could be d u e to the existence o f a finite r a n g e o f dissociation constants n e a r the a s s u m e d pKa. M e a s u r e m e n t s below p H 3.9 could help to clarify this but u n f o r t u n a t e l y m o r e acidic solutions begin to cause very large depolarizations a n d the decreases in m e m b r a n e conductances t e n d to be poorly reversible.
Effects of pH on Membrane Surface Charge In addition to d e c r e a s i n g the m a x i m u m s o d i u m a n d p o t a s s i u m c o n d u c t a n c e s , acidic solutions also alter their voltage d e p e n d e n c e . H o w e v e r , in this case the d i f f e r e n c e s b e t w e e n the effects o f p H on GNa a n d GK is striking. Fig. 5 shows n o r m a l i z e d c o n d u c t a n c e - v o l t a g e data for a typical e x p e r i m e n t in which the p H
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was r e d u c e d f r o m 7.8 to 4.2. GNa(V) is shifted by only 4 mV in the depolarized direction, while the shift in GK(V) is a p p r o x i m a t e l y 24 mV (note the d i f f e r e n c e in scale on the ordinate). T h e presence o f two points at each potential for the p H 7.8 solution is d u e to the fact that we have shown c o m p u t a t i o n s for the bracketing runs to indicate the m a g n i t u d e o f e x p e r i m e n t a l uncertainty.
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FIGURE 3. Peak inward transient and steady-state current-voltage relations for an axon at pH 7.8 and 5.5. Data were leak corrected assuming a constant leak conductance equal to that measured using a 100-mV hyperpolarizing clamp step. Solid symbols were obtained both before and after exposure to the acidic solution. Holding potential was -65 mV. Temperature 5°C. Fig. 6 shows voltage shift data pooled f r o m eight e x p e r i m e n t s . T h e s o d i u m c o n d u c t a n c e is relatively insensitive to p H , shifting by little m o r e than 1 mV per p H unit. In contrast, the potassium c o n d u c t a n c e is shifted by 4 - m V / p H unit in the r a n g e 6.5-10.2 a n d by 9 - m V / p H unit below p H 6.5. This d i f f e r e n c e in the p H sensitivity Of GK(V) is clearly outside the limits o f e x p e r i m e n t a l e r r o r , and is similar to e x p e r i m e n t s o f Hille (1968) and D r o u i n and N e u m c k e (1974) o n the s o d i u m c o n d u c t a n c e in f r o g nerve.
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FIGURE 5. Relative sodium and potassium conductances as a function of membrane potential at pH 7.8 and 4.2. U n n o r m a l i z e d values of GNa(V) and G~(V) were calculated by the relation G~(V) = I~(V - EO and were subsequently divided by the m a x i m u m conductances. Note the difference in scale Oil the voltage axis. The solid line was drawn by eye to fit the data at pH 7.8 (solid symbols) before and after exposure to acidic conditions, and then translated by 4 and 24 mV to fit the data for GNa(V) and Gz(V) at pH 4.2. Holding potential - 7 0 mV. T e m p e r a t u r e 5°C.
C , L . SCHAUF AND F . A . DAVIS
191
pH Effects on Myxicola Axons
We should point out that the potentials at which the sodium and potassium conductances reach their h a l f - m a x i m u m values, Vt Naand Vi E, are quite consistent f r o m axon to axon u n d e r constant e x p e r i m e n t a l conditions. For 10 axons at p H 7.8, V½N* averaged - 2 1 . 3 -+ 2.4 mV and Vt K was - 3 . 4 -+ 2.2 mV ( - SEM). T h e s e results suggest that a l t h o u g h the surface charge densities in the vicinity o f the sodium and potassium channels are c o m p a r a b l e at p H 7.8 (Schauf, 1975), the charges n e a r the s o d i u m channel are not very titratable over the r a n g e 3 . 9 10.2, while those n e a r the potassium channel titrate readily. In o r d e r to provide 30
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f u r t h e r c o n f i r m a t i o n o f this we a t t e m p t e d to use changes in [Ca ++] to estimate m e m b r a n e surface c h a r g e density u n d e r acidic conditions in three e x p e r i m e n t s . Translations in GNa(V) and GK(V) p r o d u c e d by increases in [Ca ++] f r o m 10 to 100 mM at p H 7.8 a g r e e d with the calculations based o n the surface c h a r g e density o f - 0 . 0 1 3 charges/A 2 d e t e r m i n e d previously in m o r e extensive experiments (Schauf, 1975). W h e n the e x p e r i m e n t was r e p e a t e d at p H 5.0, GNa(V) was shifted by a m o u n t s only slightly smaller than the shift at p H 7.8. H o w e v e r , the translation Of GK(V) was 5 - 6 mV smaller than that m e a s u r e d at p H 7.8. T h e data are not sufficiently extensive to provide an accurate value o f surface c h a r g e density, but assuming that there is n o binding o f Ca ++ (Schauf, 1975), the surface charge in the vicinity o f the K + channel seems to have been r e d u c e d to approximately 0.007 charges/A 2 at p H 5.0. This value is quite close to that which could be obtained f r o m the calculated surface potential at 60 m M divalent ion concentration, p H 7.8, and the assumption that the data in Fig. 6 simply r e p r e s e n t changes in this surface potential. T h u s the entire set o f data seems completely selfconsistent.
Effects of pH on Membrane Rate Constants Figs. 7 and 8 illustrate the effects o f acidic solutions on the time to peak s o d i u m c u r r e n t and time to h a l f - m a x i m u m potassium c u r r e n t , respectively. In the
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FIGURE 7. Time to peak inward current as a function of m e m b r a n e potential in ASW at pH 7.8 and 5.5. T h e solid symbols were obtained d u r i n g several bracketing runs at pH 7.8 and indicate the magnitude of the experimental error. Holding potential - 6 5 mV. T e m p e r a t u r e 5°C.
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FIGURE 8. T i m e to h a l f - m a x i m u m steady-state c u r r e n t as a function of m e m b r a n e potential at pH 7.8 and pH 4.6. Measurements were made in the presence of 10 -6 M T T X . T h e solid symbols were obtained d u r i n g bracketing runs at p H 7.8. Holding potential - 6 5 mV. T e m p e r a t u r e 5°C.
C. L. SCHAUFANDF. A. DAvis pHEffectsonMyxicolaAxons
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e x p e r i m e n t shown in Fig. 7 the shift in GNa(V) was only 1 mV, nevertheless t h e r e are substantial increases in time to peak. T h e data are also not particularly well described by a simple voltage shift, r a t h e r the effect is small for negative potentials, and becomes appreciable b e y o n d 0 mV. T h e effects on the potassium kinetics are even m o r e striking where for this axon the shift in GK(V) was 18 mV. For positive potentials t h e r e is at least a shift of 40 mV in the kinetics, but again the effect is not as large at negative potentials so that a simple translation c a n n o t fit all the data. (Again note the duplication o f data at p H 7.8 as an indication o f e x p e r i m e n t a l error.) T h e s e data resemble the behavior r e p o r t e d by S h r a g e r (1974) on crayfish axons, t h o u g h detailed c o m p a r i s o n is difficult, and suggests a n o t h e r , m o r e c o m p l e x , effect o f p H on excitable channels.
Effects of pH on Sodium Inactivation In several e x p e r i m e n t s steady-state sodium inactivation curves were m e a s u r e d using large test pulses. Protocols and p r o c e d u r e s were similar to those described elsewhere (Goldman and Schauf, 1972). W h e n the axons were exposed to solutions with a p H in the range 4.5-5.0, no significant effect was seen. T h e steady-state inactivation curve was translated in the depolarizated direction by 1 4 mV, a m o u n t s c o m p a r a b l e to the m a g n i t u d e o f the p H effect on GNa(V). DISCUSSION
In Myxicola axons r e d u c i n g the solution pH f r o m neutrality to acidic w!,_~_es has t h r e e d i f f e r e n t effects. T h e m a x i m u m conductances for both the Na + a n d K + channels is r e d u c e d in a m a n n e r nearly consistent with the titration of a single weak acid (pKa's o f 4.8 and 4.4 for GNa and GK, respectively). T h e m e m b r a n e surface charge density in the vicinity o f the K + channel is r e d u c e d , but its value n e a r the Na + channel remains relatively constant. Finally, t h e r e are significant effects o f acidic conditions on the activation processes in both channels b e y o n d those expected on the basis o f the alteration in surface potential. Certain aspects o f this behavior are seen in o t h e r systems. T h e relatively large effect of p H on the rate o f potassium activation c o m p a r e d to the effect on GK(V) is similar to Shrager's (1974) findings using crayfish. Although the pKa for the inhibition o f GK is larger (approximately 6.3), the shift o f GK(V) o f 25 mV for a r e d u c t i o n in p H f r o m 7.5 to 5.8 is not very m u c h larger than that seen in Myxicola over the p H r a n g e 4 - 6 . Myxicola differs f r o m crayfish primarily in the absolute value of the pKa for potassium, and in the high sensitivity o f G~a to acidic conditions. In myelinated n e r v e (Hille, 1968, 1973; D r o u i n and T h e , 1969; Mozhayeva and N a u m o v , 1970, 1972; Woodhull, 1973; D r o u i n and N e u m c k e , 1974) the s o d i u m c o n d u c t a n c e is also m o r e sensitive to acidic conditions than GK. T h e pKa for GNa is generally f o u n d to be 5.2 (but see D r o u i n and N e u m c k e , 1974), slightly h i g h e r than the value in Myxicola, while the pK~ for GK is quite close to the value o f 4.4 d e t e r m i n e d in these e x p e r i m e n t s . W h e r e the systems differ, however, is in the way in which the voltage d e p e n d e n c e o f the ionic conductances d e p e n d s on p H . In n o d e both the s o d i u m (Hille, 1968; D r o u i n and T h e , 1969; D r o u i n and N e u m c k e , 1974) and potassium (Mozhayeva and Naurrmv, 1970, 1972) conduct-
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ances are shifted in acidic solutions, whereas in Myxicola GNAW) is relatively insensitive to p H . It is interesting to observe that the relative effects o f p H on GNa(V) and GK(V) may differ for R. pipiens (HiUe, 1968) and R. esculenta (Drouin and T h e , 1969). Finally, Mozhayeva and N a u m o v (1970, 1972) r e p o r t that Gz(V) in f r o g nerve exhibits large shifts consistent with there being a second titratable g r o u p with a pKa near 9.2. No evidence for the presence of a specific titratable g r o u p with pKa < 10.2 can be f o u n d in Myxicola. Woodhull (1973) has observed in n o d e yet a n o t h e r effect o f acidic solutions. At a p H o f 5, GNa(V) was clearly c h a n g e d in shape f r o m that at p H 7 in that it did not quickly reach a plateau at positive potentials, but showed a c o n t i n u i n g , g r a d u a l increase. This was i n t e r p r e t e d as a v o l t a g e - d e p e n d e n t block o f Na + channels by H +. Such behavior does not seem to consistently exist in Myxicola axons. In Fig. 5, for example, the slight deviation of the GNa(V) curve at p H 4.2 f r o m a perfect translation is m u c h less than in W o o d h u l r s data, a n d was not clearly seen in m a n y cases. H o w e v e r , the data are certainly not c o m p e l l i n g since the major effect in n o d e occurs at potentials where m e m b r a n e potassium currents are large, and we did not carry out e x p e r i m e n t s using T E A + internally. T h e pKa for a carboxyl side chain in a protein molecule is given by T a n f o r d (1961) as 4.7. Presumably carboxyl g r o u p s in slightly different local environments would have dissociation constants which differed slightly from one another, possibly i n t r o d u c i n g some "splay" in the titration curve. T h e data in Myxicola would certainly be consistent with the presence o f such a dissociable g r o u p in the sodium c h a n n e l as has been a r g u e d for the n o d e o f Ranvier (Hille, 1975). We are grateful to Ms. Barbara J. Reed for expert technical assistance. This work was supported by the Morris Multiple Sclerosis Research Fund. Charles L. Schauf is the recipient of a Research Career Development Award from the National Institutes of Health (1 KO4 NS 00004-01).
Receivedfor publication 9 June 1975. R E F E R E N C E S
BINSTOCK, L., and L. GOLDMAN. 1969. Current and voltage clamped studies on Myxicola giant axons: effect of tetrodotoxin. J. Gen. Physiol. 54:730. DROUIN, H., and R. ThE. 1969. The effect of reducing extracellular pH on the membrane currents of the Ranvier node. Pfluegers Arch. Eur. J. Physiol. 313:80. DROUIN, H., and B. Nr'.VMCKr. 1974. Specific and unspecific charges at the sodium channels of the nerve membrane. Pfluegers Arch. Eur. J. Physiol. 351:207. EHRENSTEIN, G., and H. M. FISHMAN. 1971. Evidence against hydrogen-calcium competition model for activation of electrically excitable membranes. Nat. New Biol. 233:16. GOLDMAN, L., and C. L. SCHACV. 1972. Inactivation of the sodium current in Myxicola giant axons: evidence for coupling to the activation process.J. Gen. Physiol. 59:659. GOLDMAN,L., and C. L. SCHAUV. 1973. Quantitative description of sodium and potassium current and computed action potentials in Myxicola giant axons.J. Gen. Physiol. 61:361. HILLE, B. 1968. Charges and potentials at the nerve surface. Divalent ions and pH.J. Gen. Physiol. 51:221.
C. L. SCHAUFAND F. A. DAVIS pH Effects on MyxieotaAxons
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HtLLE, B. 1973. Potassium channels in myelinated nerve: selective permeability to small cations.J. Gen. Physiol. 61:669. HILLE, B. 1975. An essential ionized acid g r o u p in sodium channels. Fed. Proc. 34:1318. MOZHAVrVA, G. N., and A. P. NAUMOV. 1970. Effect o f surface charge on the steady-state potassium conductance of nodal m e m b r a n e . Nature (Lond.). 228:164. MOZr~AYEVA, G. N., and A. P. NAUMOV. 1972. Effect of the surface charge on the steady potassium conductivity o f the m e m b r a n e o f a node o f Ranvier. 1. Change in p H o f external solution. Biophysics. 17:644. SHRA~ER, P. 1974. Ionic conductance changes in voltage c l a m p e d crayfish axons at low p H . J . Gen. Physiol. 64:666. STILLMAN,I. M., D. L. GILBERT,and R . J . LtpxcKv. 1971. Effect o f external pH u p o n the voltage d e p e n d e n t currents o f the squid giant axon. Biophys. J. 11:55a. SCHAVF, C. 1973. T e m p e r a t u r e d e p e n d e n c e o f the ionic current kinetics of Myxicola giant a x o n s . J . Physiol. (Lond.), 235:197. SCHAUF, C. 1975. T h e interactions of calcium with Myxicola giant axons and a description in terms o f a simple surface charge m o d e I . J . Physiol. (Lond.). 248:613. TANFORO, C. 1961. Physical Chemistry of Macromolecules. J o h n Wiley and Sons, Inc., New York. WOODHULL, A. M. 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61:687.
a n d F. A. D A V I S
From the Departments of Neurological Sciences and Physiology, Rush Medical College, Chicago, Illinois 60612
M y x i c o l a giant axons were studied using standard voltage-clamp techniques in solutions whose pH values ranged from 3.9 to 10.2. Buffer concentrations of 50 mM or greater were necessary to demonstrate the full effect of pH. In acidic solutions the axon underwent a variable depolarization, and both the sodium and potassium conductances were reversibly depressed with approximate pKa's of 4.8 and 4.4, respectively. The voltage dependence of G~a was only slightly altered by acidic conditions, whereas there occurred large shifts in GK along the voltage axis consistent with a substantial decrease in net negative surface charge in the vicinity of the K + channels. The sodium and potassium activation rate constants were decreased by acidic conditions, but the results could not be described as a simple translation along the voltage axis. A B S ' r R A C "r
INTRODUCTION Alteration o f the e x t e r n a l p H basically has two distinct effects on m e m b r a n e p a r a m e t e r s . Various r e p o r t s (Hille, 1968, 1973; D r o u i n a n d T h e , 1969; Mozhayeva a n d N a u m o v , 1970, 1972; E h r e n s t e i n a n d F i s h m a n , 1971; Stillman et al., 1971; W o o d h u l l , 1973; D r o u i n a n d N e u m c k e , 1974; S h r a g e r , 1974) have shown that u n d e r acidic conditions b o t h the m a x i m u m s o d i u m a n d p o t a s s i u m c o n d u c t ances are reversibly d e p r e s s e d in a m a n n e r m o r e or less consistent with the titration o f a weak acid. In f r o g n e r v e t h e r e is general a g r e e m e n t that the pKa f o r the s o d i u m c o n d u c t a n c e is a r o u n d 5.2, whereas for p o t a s s i u m the pKa is n e a r 4.5. In squid the pK~ f o r the s o d i u m c h a n n e l was f o u n d to be 6.5, while GK was nearly u n a f f e c t e d by p H values a b o v e 5 (Stillman et al., 1971). H o w e v e r , in crayfish S h r a g e r (1974) f o u n d a pKa o f 6.3 for the p o t a s s i u m c o n d u c t a n c e , a n d c o m m e n t e d that the s o d i u m c o n d u c t a n c e was relatively less sensitive to c h a n g e s in p H . In addition to this effect on c h a n n e l c o n d u c t a n c e , a r e d u c t i o n in e x t e r n a l p H also has a m a r k e d effect o n the voltage d e p e n d e n c e o f a variety o f m e m b r a n e p a r a m e t e r s , a n d h e r e also the results d e p e n d on the p r e p a r a t i o n e m p l o y e d . I n f r o g n e r v e b o t h the s o d i u m a n d p o t a s s i u m c o n d u c t a n c e - v o l t a g e relations are shifted in the d e p o l a r i z e d direction as the solution b e c o m e s increasingly acidic, a l t h o u g h the effect m a y be s o m e w h a t g r e a t e r for the p o t a s s i u m c h a n n e l (Hille, THE JOURNALOF GENERALPHYSIOLOGY VOLUME67, 1976 ' pages 185-195 "
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1968; D r o u i n a n d T h e , 1969). T h e r a t e c o n s t a n t s g e n e r a l l y a p p e a r to b e s h i f t e d by a m o u n t s c o m p a r a b l e to t h e c o n d u c t a n c e s . I n c r a y f i s h a x o n s , h o w e v e r , t h e e f f e c t o f p H o n t h e r a t e c o n s t a n t f o r p o t a s s i u m a c t i v a t i o n is m u c h l a r g e r t h a n t h e effect on the conductance, and combined with a relative insensitivity of the s o d i u m c h a n n e l to p H , t h i s r e s u l t s in a n e a r l y c o m p l e t e t e m p o r a l s e p a r a t i o n o f t h e s o d i u m a n d p o t a s s i u m c u r r e n t s ( S h r a g e r , 1974). D e t a i l e d d a t a h a v e n o t b e e n reported for squid axons. B e c a u s e o f t h e s e v a r i a t i o n s in r e s p o n s i v e n e s s to p H f r o m o n e s y s t e m t o a n o t h e r , a n d t h e e x i s t e n c e o f c o m p l e t e d a t a f o r b o t h c h a n n e l s o n l y in n o d e , we felt it a p p r o p r i a t e to c a r r y o u t s i m i l a r s t u d i e s in v o l t a g e - c l a m p e d Myxicola g i a n t a x o n s . I n a d d i t i o n , s u c h s t u d i e s t e n d to c o m p l e m e n t t h e p r e v i o u s i n v e s t i g a t i o n o f m e m b r a n e s u r f a c e c h a r g e in this p r e p a r a t i o n ( S c h a u f , 1975). METHODS
Myxicola giant
axons were voltage clamped and the resulting data analyzed by methods described in detail elsewhere (Binstock and G o l d m a n , 1969; G o l d m a n and Schauf, 1972, 1973; Schauf, 1973, 1975). Compensated feedback was used in all experiments. T h e reference artificial seawater (ASW) solution had the composition: 430 mM NaCI, 10 mM KCi, 10 mM CaCi2, 50 mM MgCi2. In a few cases Mg ++ free seawater having various Ca ++ concentrations was used. T e m p e r a t u r e was maintained at 5.0 - 0.5°C. In the initial experiments 5 mM Tris maleate had been used as the buffer system. At this b u f f e r i n g capacity no changes in m e m b r a n e parameters could be observed after changes in solution p H . A nearly comparable insensitivity was f o u n d at 10 mM buffer. Only when the concentration o f Tris maleate (or Tris acetate in experiments at the most acidic p H values) was raised to 50 mM was the effect o f p H readily a p p a r e n t . A f u r t h e r increase in buffer concentration to 100 mM proved no m o r e effective than 50 mM. T h u s all the data r e p o r t e d here were obtained using 50 mM Tris maleate or Tris acetate with the p H adjusted by addition of HCI or N a O H just before an e x p e r i m e n t , and r e m e a s u r e d immediately after its use on an axon. T h e high t e m p e r a t u r e coefficient of Tris was taken into account. It should be pointed out that there was no detectable effect on m e m b r a n e parameters when the b u f f e r concentration was increased from 5 to 50 mM at the s t a n d a r d p H o f 7.8, only when buffer concentration was increased u n d e r acidic conditions. In the majority o f cases tetrodotoxin (TTX) was not routinely used to accurately obtain n o n s o d i u m currents. Rather peak transient and steady-state currents were corrected for the leak conductance measured d u r i n g a 100-mV hyperpolarization. However, in those experiments involving measurements o f the rate o f potassium activation, or steady-state sodium inactivation, T T X (10 -6 M) was used. In addition, a few experiments were done with T T X to ensure that there was in fact no significant difference in the conductance shifts measured using either method. A solution of ASW at a p H o f 7.8 -+ 0.05 served as the control for all measurements. T h e effects o f p H were usually complete within a few minutes, but we normally allowed the system to equilibrate for 15 min before r e c o r d i n g data. Each e x p e r i m e n t at an acidic or basic p H was bracketed by runs at the reference p H of 7.8. T h e m a x i m u m sodium a n d potassium conductances, as well as the half-maximal potentials (Vi sa and Vt K) were averaged for the bracketing runs at 7.8, and the relative conductance or relative voltage shift calculated using these averaged values. Axons in which there were very large differences between the bracketing values were assumed to have d e t e r i o r a t e d d u r i n g the course of the e x p e r i m e n t a n d the data were not used.
C. L. SCHAUFAND [7. A. DAVXS pHEffects on MyxicolaAxons
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RESULTS
Fig. 1 illustrates the effect o f an acidic solution on the m e m b r a n e c u r r e n t s in
Myxicola. R e d u c i n g the p H f r o m 7.8 to 4.6 causes an inhibition o f b o t h the p e a k transient a n d steady-state c u r r e n t s , t h o u g h the f o r m e r is slightly m o r e sensitive. T h e r e is a large decrease in the rate o f activation o f the p o t a s s i u m c u r r e n t which is b e t t e r s h o w n in Fig. 2 w h e r e 10 -e M T T X has b e e n used to eliminate m e m b r a n e s o d i u m c u r r e n t . A l t h o u g h it m a y be difficult to discern f r o m the r e c o r d s in Fig. 1, the time to p e a k i n w a r d c u r r e n t is significantly increased as well. H o w e v e r , in contrast to the results o f S h r a g e r (1974) o n crayfish axons, the s o d i u m a n d p o t a s s i u m c u r r e n t s do not b e c o m e t e m p o r a l l y s e p a r a t e d u n d e r acidic conditions. M e m b r a n e c u r r e n t - v o l t a g e relations at n o r m a l a n d acid p H are shown in Fig. 3, a f t e r correction f o r a linear leak. T h e s o d i u m l ( V ) c u r v e is d e c r e a s e d with little sign o f a voltage shift a n d with n o significant c h a n g e in the s o d i u m e q u i l i b r i u m potential (ENa). In contrast, the p o t a s s i u m I(V) shows a m a r k e d translation in the d e p o l a r i z e d direction a l o n g the voltage axis. In b o t h cases reversibility is excellent. It should be n o t e d that in Myxicola a x o n s a r e d u c t i o n o f the solution p H below 6.0 causes an a p p r e c i a b l e m e m b r a n e d e p o l a r i z a t i o n (range 4 - 1 2 mV), which is h o w e v e r c o m p l e t e l y reversible. T h e data show a large a m o u n t o f scatter, howe v e r , a n d it is impossible to d e m o n s t r a t e a m o n o t o n i c p H d e p e n d e n c e o f m e m b r a n e potential.
FIGURE 1. Voltage-clamp currents recorded in ASW at a pH 7.8 (top) and 4.6 (bottom) in response to depolarizations of 40, 50, 60, 70, 80, 90, and 100 mV from a holding potential of - 6 0 mV. Current and time scales are 0.3 mA/cm 2 and 2.0 ms, respectively. Temperature 5°C.
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FIGURE 2. Voltage-clamp current recorded in ASW + 10-8 M T T X at a pH 7.8 (top) and 4.6 (bottom) in response to depolarizations of 20, 40, 60, 80,100,120, and 140 mV from a holding potential of -65 mV. Current and time scales are 0.3 mA/cm2 and 2.0 ms, respectively. Temperature 5°C.
Effects of pH on Channel Conductance I n Fig. 4 we have plotted the m a x i m u m s o d i u m a n d p o t a s s i u m c o n d u c t a n c e s as a function o f solution p H , n o r m a l i z e d to the c o n d u c t a n c e s o b s e r v e d at the r e f e r ence p H o f 7.8. Data are only shown over the r a n g e 3.9-7.8, but were o b t a i n e d u p to a p H o f 10.2. W h e n we a v e r a g e d the values o f relative c o n d u c t a n c e at p H values above 7.8 we o b t a i n e d 1.01 -+ 0.03 for potassium (-+ SEM) a n d 1.03 - 0.04 for s o d i u m with no evidence for a n y systematic d e p e n d e n c e on p H (six experiments). T h e solid lines are calculated f r o m the dissociation o f a weak acid with a pKa o f 4.8 for G N a a n d 4.4 for GK. A l t h o u g h the solid lines m o r e or less describe the p H d e p e n d e n c e o f the m a x i m u m channel c o n d u c t a n c e , t h e r e seems to be s o m e systematic deviation. Points at p H values l a r g e r t h a n the pKa are below the line, while those at a p H m o r e acidic t h a n the pK~ are above. T h i s deviation could be d u e to the existence o f a finite r a n g e o f dissociation constants n e a r the a s s u m e d pKa. M e a s u r e m e n t s below p H 3.9 could help to clarify this but u n f o r t u n a t e l y m o r e acidic solutions begin to cause very large depolarizations a n d the decreases in m e m b r a n e conductances t e n d to be poorly reversible.
Effects of pH on Membrane Surface Charge In addition to d e c r e a s i n g the m a x i m u m s o d i u m a n d p o t a s s i u m c o n d u c t a n c e s , acidic solutions also alter their voltage d e p e n d e n c e . H o w e v e r , in this case the d i f f e r e n c e s b e t w e e n the effects o f p H on GNa a n d GK is striking. Fig. 5 shows n o r m a l i z e d c o n d u c t a n c e - v o l t a g e data for a typical e x p e r i m e n t in which the p H
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C. L. SCHAUF AND F. A. DAVIS pHEffects on Myxicola Axons
was r e d u c e d f r o m 7.8 to 4.2. GNa(V) is shifted by only 4 mV in the depolarized direction, while the shift in GK(V) is a p p r o x i m a t e l y 24 mV (note the d i f f e r e n c e in scale on the ordinate). T h e presence o f two points at each potential for the p H 7.8 solution is d u e to the fact that we have shown c o m p u t a t i o n s for the bracketing runs to indicate the m a g n i t u d e o f e x p e r i m e n t a l uncertainty.
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FIGURE 3. Peak inward transient and steady-state current-voltage relations for an axon at pH 7.8 and 5.5. Data were leak corrected assuming a constant leak conductance equal to that measured using a 100-mV hyperpolarizing clamp step. Solid symbols were obtained both before and after exposure to the acidic solution. Holding potential was -65 mV. Temperature 5°C. Fig. 6 shows voltage shift data pooled f r o m eight e x p e r i m e n t s . T h e s o d i u m c o n d u c t a n c e is relatively insensitive to p H , shifting by little m o r e than 1 mV per p H unit. In contrast, the potassium c o n d u c t a n c e is shifted by 4 - m V / p H unit in the r a n g e 6.5-10.2 a n d by 9 - m V / p H unit below p H 6.5. This d i f f e r e n c e in the p H sensitivity Of GK(V) is clearly outside the limits o f e x p e r i m e n t a l e r r o r , and is similar to e x p e r i m e n t s o f Hille (1968) and D r o u i n and N e u m c k e (1974) o n the s o d i u m c o n d u c t a n c e in f r o g nerve.
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Relative sodium and potassium conductances as a function of solution pH. Unnormalized GNa(V) and Gz(V) curves similar to those shown in Fig. 5 were constructed for the data in each solution and the m a x i m u m values determined. T h e conductances at a particular pH are expressed as a fraction of the average of the m a x i m u m conductances at pH 7.8 before and after exposure to acidic solutions. T h e solid lines are calculated assuming the dissociation of a single weak acid with the indicated pKa. Data pooled from eight axons,
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FIGURE 5. Relative sodium and potassium conductances as a function of membrane potential at pH 7.8 and 4.2. U n n o r m a l i z e d values of GNa(V) and G~(V) were calculated by the relation G~(V) = I~(V - EO and were subsequently divided by the m a x i m u m conductances. Note the difference in scale Oil the voltage axis. The solid line was drawn by eye to fit the data at pH 7.8 (solid symbols) before and after exposure to acidic conditions, and then translated by 4 and 24 mV to fit the data for GNa(V) and Gz(V) at pH 4.2. Holding potential - 7 0 mV. T e m p e r a t u r e 5°C.
C , L . SCHAUF AND F . A . DAVIS
191
pH Effects on Myxicola Axons
We should point out that the potentials at which the sodium and potassium conductances reach their h a l f - m a x i m u m values, Vt Naand Vi E, are quite consistent f r o m axon to axon u n d e r constant e x p e r i m e n t a l conditions. For 10 axons at p H 7.8, V½N* averaged - 2 1 . 3 -+ 2.4 mV and Vt K was - 3 . 4 -+ 2.2 mV ( - SEM). T h e s e results suggest that a l t h o u g h the surface charge densities in the vicinity o f the sodium and potassium channels are c o m p a r a b l e at p H 7.8 (Schauf, 1975), the charges n e a r the s o d i u m channel are not very titratable over the r a n g e 3 . 9 10.2, while those n e a r the potassium channel titrate readily. In o r d e r to provide 30
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f u r t h e r c o n f i r m a t i o n o f this we a t t e m p t e d to use changes in [Ca ++] to estimate m e m b r a n e surface c h a r g e density u n d e r acidic conditions in three e x p e r i m e n t s . Translations in GNa(V) and GK(V) p r o d u c e d by increases in [Ca ++] f r o m 10 to 100 mM at p H 7.8 a g r e e d with the calculations based o n the surface c h a r g e density o f - 0 . 0 1 3 charges/A 2 d e t e r m i n e d previously in m o r e extensive experiments (Schauf, 1975). W h e n the e x p e r i m e n t was r e p e a t e d at p H 5.0, GNa(V) was shifted by a m o u n t s only slightly smaller than the shift at p H 7.8. H o w e v e r , the translation Of GK(V) was 5 - 6 mV smaller than that m e a s u r e d at p H 7.8. T h e data are not sufficiently extensive to provide an accurate value o f surface c h a r g e density, but assuming that there is n o binding o f Ca ++ (Schauf, 1975), the surface charge in the vicinity o f the K + channel seems to have been r e d u c e d to approximately 0.007 charges/A 2 at p H 5.0. This value is quite close to that which could be obtained f r o m the calculated surface potential at 60 m M divalent ion concentration, p H 7.8, and the assumption that the data in Fig. 6 simply r e p r e s e n t changes in this surface potential. T h u s the entire set o f data seems completely selfconsistent.
Effects of pH on Membrane Rate Constants Figs. 7 and 8 illustrate the effects o f acidic solutions on the time to peak s o d i u m c u r r e n t and time to h a l f - m a x i m u m potassium c u r r e n t , respectively. In the
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MEMBRANE POTENTIAL (mY)
FIGURE 8. T i m e to h a l f - m a x i m u m steady-state c u r r e n t as a function of m e m b r a n e potential at pH 7.8 and pH 4.6. Measurements were made in the presence of 10 -6 M T T X . T h e solid symbols were obtained d u r i n g bracketing runs at p H 7.8. Holding potential - 6 5 mV. T e m p e r a t u r e 5°C.
C. L. SCHAUFANDF. A. DAvis pHEffectsonMyxicolaAxons
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e x p e r i m e n t shown in Fig. 7 the shift in GNa(V) was only 1 mV, nevertheless t h e r e are substantial increases in time to peak. T h e data are also not particularly well described by a simple voltage shift, r a t h e r the effect is small for negative potentials, and becomes appreciable b e y o n d 0 mV. T h e effects on the potassium kinetics are even m o r e striking where for this axon the shift in GK(V) was 18 mV. For positive potentials t h e r e is at least a shift of 40 mV in the kinetics, but again the effect is not as large at negative potentials so that a simple translation c a n n o t fit all the data. (Again note the duplication o f data at p H 7.8 as an indication o f e x p e r i m e n t a l error.) T h e s e data resemble the behavior r e p o r t e d by S h r a g e r (1974) on crayfish axons, t h o u g h detailed c o m p a r i s o n is difficult, and suggests a n o t h e r , m o r e c o m p l e x , effect o f p H on excitable channels.
Effects of pH on Sodium Inactivation In several e x p e r i m e n t s steady-state sodium inactivation curves were m e a s u r e d using large test pulses. Protocols and p r o c e d u r e s were similar to those described elsewhere (Goldman and Schauf, 1972). W h e n the axons were exposed to solutions with a p H in the range 4.5-5.0, no significant effect was seen. T h e steady-state inactivation curve was translated in the depolarizated direction by 1 4 mV, a m o u n t s c o m p a r a b l e to the m a g n i t u d e o f the p H effect on GNa(V). DISCUSSION
In Myxicola axons r e d u c i n g the solution pH f r o m neutrality to acidic w!,_~_es has t h r e e d i f f e r e n t effects. T h e m a x i m u m conductances for both the Na + a n d K + channels is r e d u c e d in a m a n n e r nearly consistent with the titration of a single weak acid (pKa's o f 4.8 and 4.4 for GNa and GK, respectively). T h e m e m b r a n e surface charge density in the vicinity o f the K + channel is r e d u c e d , but its value n e a r the Na + channel remains relatively constant. Finally, t h e r e are significant effects o f acidic conditions on the activation processes in both channels b e y o n d those expected on the basis o f the alteration in surface potential. Certain aspects o f this behavior are seen in o t h e r systems. T h e relatively large effect of p H on the rate o f potassium activation c o m p a r e d to the effect on GK(V) is similar to Shrager's (1974) findings using crayfish. Although the pKa for the inhibition o f GK is larger (approximately 6.3), the shift o f GK(V) o f 25 mV for a r e d u c t i o n in p H f r o m 7.5 to 5.8 is not very m u c h larger than that seen in Myxicola over the p H r a n g e 4 - 6 . Myxicola differs f r o m crayfish primarily in the absolute value of the pKa for potassium, and in the high sensitivity o f G~a to acidic conditions. In myelinated n e r v e (Hille, 1968, 1973; D r o u i n and T h e , 1969; Mozhayeva and N a u m o v , 1970, 1972; Woodhull, 1973; D r o u i n and N e u m c k e , 1974) the s o d i u m c o n d u c t a n c e is also m o r e sensitive to acidic conditions than GK. T h e pKa for GNa is generally f o u n d to be 5.2 (but see D r o u i n and N e u m c k e , 1974), slightly h i g h e r than the value in Myxicola, while the pK~ for GK is quite close to the value o f 4.4 d e t e r m i n e d in these e x p e r i m e n t s . W h e r e the systems differ, however, is in the way in which the voltage d e p e n d e n c e o f the ionic conductances d e p e n d s on p H . In n o d e both the s o d i u m (Hille, 1968; D r o u i n and T h e , 1969; D r o u i n and N e u m c k e , 1974) and potassium (Mozhayeva and Naurrmv, 1970, 1972) conduct-
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. 1976
ances are shifted in acidic solutions, whereas in Myxicola GNAW) is relatively insensitive to p H . It is interesting to observe that the relative effects o f p H on GNa(V) and GK(V) may differ for R. pipiens (HiUe, 1968) and R. esculenta (Drouin and T h e , 1969). Finally, Mozhayeva and N a u m o v (1970, 1972) r e p o r t that Gz(V) in f r o g nerve exhibits large shifts consistent with there being a second titratable g r o u p with a pKa near 9.2. No evidence for the presence of a specific titratable g r o u p with pKa < 10.2 can be f o u n d in Myxicola. Woodhull (1973) has observed in n o d e yet a n o t h e r effect o f acidic solutions. At a p H o f 5, GNa(V) was clearly c h a n g e d in shape f r o m that at p H 7 in that it did not quickly reach a plateau at positive potentials, but showed a c o n t i n u i n g , g r a d u a l increase. This was i n t e r p r e t e d as a v o l t a g e - d e p e n d e n t block o f Na + channels by H +. Such behavior does not seem to consistently exist in Myxicola axons. In Fig. 5, for example, the slight deviation of the GNa(V) curve at p H 4.2 f r o m a perfect translation is m u c h less than in W o o d h u l r s data, a n d was not clearly seen in m a n y cases. H o w e v e r , the data are certainly not c o m p e l l i n g since the major effect in n o d e occurs at potentials where m e m b r a n e potassium currents are large, and we did not carry out e x p e r i m e n t s using T E A + internally. T h e pKa for a carboxyl side chain in a protein molecule is given by T a n f o r d (1961) as 4.7. Presumably carboxyl g r o u p s in slightly different local environments would have dissociation constants which differed slightly from one another, possibly i n t r o d u c i n g some "splay" in the titration curve. T h e data in Myxicola would certainly be consistent with the presence o f such a dissociable g r o u p in the sodium c h a n n e l as has been a r g u e d for the n o d e o f Ranvier (Hille, 1975). We are grateful to Ms. Barbara J. Reed for expert technical assistance. This work was supported by the Morris Multiple Sclerosis Research Fund. Charles L. Schauf is the recipient of a Research Career Development Award from the National Institutes of Health (1 KO4 NS 00004-01).
Receivedfor publication 9 June 1975. R E F E R E N C E S
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HtLLE, B. 1973. Potassium channels in myelinated nerve: selective permeability to small cations.J. Gen. Physiol. 61:669. HILLE, B. 1975. An essential ionized acid g r o u p in sodium channels. Fed. Proc. 34:1318. MOZHAVrVA, G. N., and A. P. NAUMOV. 1970. Effect o f surface charge on the steady-state potassium conductance of nodal m e m b r a n e . Nature (Lond.). 228:164. MOZr~AYEVA, G. N., and A. P. NAUMOV. 1972. Effect of the surface charge on the steady potassium conductivity o f the m e m b r a n e o f a node o f Ranvier. 1. Change in p H o f external solution. Biophysics. 17:644. SHRA~ER, P. 1974. Ionic conductance changes in voltage c l a m p e d crayfish axons at low p H . J . Gen. Physiol. 64:666. STILLMAN,I. M., D. L. GILBERT,and R . J . LtpxcKv. 1971. Effect o f external pH u p o n the voltage d e p e n d e n t currents o f the squid giant axon. Biophys. J. 11:55a. SCHAVF, C. 1973. T e m p e r a t u r e d e p e n d e n c e o f the ionic current kinetics of Myxicola giant a x o n s . J . Physiol. (Lond.), 235:197. SCHAUF, C. 1975. T h e interactions of calcium with Myxicola giant axons and a description in terms o f a simple surface charge m o d e I . J . Physiol. (Lond.). 248:613. TANFORO, C. 1961. Physical Chemistry of Macromolecules. J o h n Wiley and Sons, Inc., New York. WOODHULL, A. M. 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61:687.
