Pflfigers Arch (1986) 406:31 - 36 Pflfigers Archiv

European Journal of Physiology

�9 Springer-Vedag 1986

Voltage dependent modification of sodium channel gating with water-soluble carbodiimide G. N. Mozhayeva, A. P. Naumov, and E. D. Nosyreva

Institute of Cytology, Academy of Sciences of the USSR, Tikhoretsky ave. 4, SU-t94064 Leningrad, USSR

Abstract. Currents through sodium channels in frog myelinated fibre were measured before and after the treat- ment of the membrane with watersoluble carbodiimide (WSC). The WSC treatment produces dramatic changes both in activation and inactivation when membrane poten- tial during the treatment is held at levels from - 8 0 to - 100 mV. Both gating processes are slowed and their volt- age dependence is reduced. The effective charge of activation as measured by limiting logarithmic potential sensitivity is reduced after 10 rain of the WSC treatment by the factor of 1.66. The same WSC treatment applied at zero potential induced no change in the effective charge of activation. Other parameters of gating are changed after such treatment but to a lesser degree and in a somewhat different fashion than after the treatment at high negative potential. The results are consistent with the hypothesis that part of a mobile gating charge is presented by carboxyl groups migrating from the external surface to the interior of the channel molecule when the channel opens or inactivates.

Key words: Voltage clamp -- Nodal membrane - Sodium channel - C a r b o d i i m i d e s - Carboxyl groups - Mobile charges

Introduction

Ion conductance in electrosensitive sodium channels is controlled by two potential-dependent gating processes: activation and inactivation (Hodgkin and Huxley 1952). The potential dependence of gating is provided by the system of mobile charges in gating machinery (see e.g. Almers 1978). At present, little is known about specific components of the channel involved in gating though the functional properties of activation and inactivation have been described in detail. One reasonable approach to the problem of relationships between the specific structures and the functions in channel is a chemical modification. A number of chemical reagents and enzymes applied internally (Armstrong et al. 1973; Rojas and Rudy 1976; Eadon and Brodwick 1979; Oxford 1981) or externally (Shrager 1977; Wang 1984; Pappone and Cahalan 1984) were shown to modify or eliminate inactiva- tion of sodium channels without essentially changing activa- tion. It was not until recently that chemical modification of activation was demonstrated (Mozhayeva et al. 1984a, b). Both activation and inactivation proved to be dramatically altered following the treatment of the membrane with one of the water-soluble carbodiimides (WSC), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide HC1. WSC is known to be reactive, under certain conditions, to carboxyl groups of

Offprint requests to: G. N. Mozhayeva at the above address

proteins (Riehm and Sheraga 1966; Pdordan and Hayashida 1970; Timkovich 1977; Millett et al. 1982, 1983; Geren et al. 1984).

The most interesting effect of the WSC treatment on sodium channels was the decrease in so called limiting loga- rithmic potential sensitivity of activation (Almers 1978). This finding suggested the hypothesis that carboxyl groups accessible from the external solution are important components of the channel gating mechanism. It is conceiv- able that they serve as mobile gating particles. Then, such gating particles are expected to behave as was proposed by Gilly and Armstrong (1982), to reside on the external side of the channel molecule when the membrane potential is negative enough for the channel to be closed and to move into the depth of the channel structure as the membrane is depolarized and the channel opens or inactivates.

I f carboxyl groups are components of gating mecha- nism, i.e. if they themselves are mobile or are in contact with other mobile structures, their accessibility to the reagent should generally vary depending on the state the channel assumes.

The main objective of the present work was to examine whether the WSC effects on gating in sodium channel are state-dependent or not. For voltage-dependent channels the state dependence manifests itself in a voltage dependence. We have therefore performed two sets of experiments which differ from each other in potential during the WSC treat- ment.

Some of the results have been presented in abstract form (Mozhayeva et al. 1984c).

Material and methods

The work was done on myelinated fibres of the frog Rana ridibunda under voltage clamp conditions (Mozhayeva et al. 1977).

Membrane potential (E) was referred to the outside. Membrane currents were calibrated on the assumption that the resistance of the current-feeding internode was equal to 20 M~2. Sodium current measurements were accomplished with P/2 protocol (Mozhayeva and Naumov 1983; Mozhayeva et al. 1984a, b).

The WSC treatment resulted in a considerable inhibi- tion of the total sodium conductance. To avoid any misinterpretation of the results due to current-dependent artefacts, current measurements before the treatment were as a rule carried out with depolarizing prepulses, inactivating most of sodium conductance (gNa). This procedure has been shown before (Mozhayeva et al. 1984a) not to influence the time course of the sodium current nor the voltage dependence of fractional number of channels open at the

32

current peak. Additionally, compensation for series resis- tance (Rs) was used in all experiments, with Rs assumed to be 300 kO.

The control solution contained (in mM): 110NaC1, 2 CaCI2, 8 tetraethylammonium chloride, 10 Tris-HC1 (pH 7.5-7.6) . Fibres were cut in a solution containing 120 mM CsF.

Two water-soluble carbodiimide species were used in this work: l-ethyl-3-(3-dimethylaminopropyl)carbodiimide HC1 (EDC) and N-cyclohexyl-3-(2-morpholinyl)-ethyl carbo- diimide metho-4-toluene sulfonate (CMC), both from Serva, Heidelberg, FRG. Solutions for the treatment contained 100 mM WSC and 5 mM CaCI2; additionally, they contained 20 mM of monovalent cation (Na, K or Tris) salt to be nearly of the same osmolarity as the control solution was. pH of most solutions with WSC was adjusted to about 5.0 either by a mixture of 2-(N-morpholino)-ethan-sulfonic acid (MES) plus Tris base or by addition of small amounts of HC1. Carbodiimide solutions were prepared just before application. After the treatment the WSC solution was re- placed by the control solution in which all measurements have been done. The experiments were performed at 9 - 10 ~

Since the main goal of the present work was to reveal a difference in gating properties of the sodium channels in normal and treated membrane we chose to describe gating in terms of several simplified parameters which could be easily determined from macroscopic sodium current mea- surements.

Channel openness. The fractional number of channels open at the peak of the current (np) as a function of potential can be determined from peak current-voltage and "in- stantaneous" current-voltage relations corresponding to the constant number of open channels as the normalized ratio of peak currents value to the "instantaneous" one (Chiu 1980). This method was used completely in several experi- ments. In the remainder np was simply determined as a normalized ratio of peak current values to current values obtained by extrapolation of the linear part of peak current- voltage relations toward more negative potentials. Both methods gave the same results because current-voltage re- lations for the constant nmnber of normal or WSC modified channels are essentially linear over the voltages where the channel opening takes place (Mozhayeva et al. 1984a).

The middle part of the np(E) curve was described by the equation (Mozhayeva et al. 1980)

n p = [exp a g ( E - - Eg)]" [1 -}- exp a g ( E - - Eg)] -1 , (1)

where ag the steepness parameter (in mV-1) and Eg the potential of half-activation (in mV). np values lower than 0.1 were not used when fitting curves.

At potentials where channels just begin to open (np < 0.1) the semilogarithmic plot of np against E is presented by a straight line with the slope proportional to the so-called equivalent gating charge (Zof) (Hodgkin and Huxley 1952; Almers 1978). The displacement of this charge in the electric field is required for transition of the channel between the open and closed state ("fully" closed state) which is energetically most distant from it. Strictly speaking, the effective charge of activation is an equilibrium parameter of the gating machinery and therefore can be determined from the n p ( E ) curve only for channels without inactivation. How- ever, the shape of the voltage dependence of np has been

shown to be greatly insensitive to removal (Armstrong et al. 1973; Oxford 1981; Carbone et al. 1981; Wang 1984) or deceleration of inactivation (Mozhayeva et al. 1980) or decreasing the percentage of noninactivated channels by depolarizing prepulses (Mozhayeva et al. 1984 a). Therefore the peak conductance curve is essentially indistinguishable from the equilibrium activation curve and can be used for determination of an effective charge of activation.

Current rise was described by the simplified equation

I(t) = Ip[1 - e x p ( - t/Zp)] (2)

where Ip the peak current value and Zp the apparent time constant of activation. The rate at which current reaches the peak value depends not only on the rate constants of the activation steps but also, to a certain degree, on the inactiva- tion rate constants. This degree does not seem to be very considerable for channels in the nodal membrane. Thus, the half-time to current peak (parameter which differs from Zp only by constant factor) changed by no more than 20% after treatment of the membrane with chloramine-T resulting in a drastic inhibition of inactivation (Wang 1984).

Voltage dependence of inactivation was determined using a conventional double pulse protocol with a constant test pulse and varied prepulse 100 ms in duration. Current re- sponse to the test pulse normalized to its maximal value was taken as a measure of the fraction of noninactivated channels (h~). Control experiments showed that 100 ms duration was sufficient to reach a stady-state, boo(E) curve was described by the equation (Mozhayeva et al. 1980):

h~ = [1 + exp a h ( E - E~)] -1 (3)

where ah the steepness parameter (in mV -1) and Eh the potential of half-inactivation. This equation is not intended to describe the foot of the curve. The fit was therefore limited to potentials where h~ values were no less than 0.2.

Time course of current inactivation was described as two exponential processes with the slow (Zhs) and fast (%f) time constants.

Averaged data are presented as mean + SEM (number of experiments).

Results

Two parallel series of experiments were performed: in one series WSC was applied at normal ( - 8 0 to - 1 0 0 mV) holding potential (EH) as in previous work (Mozhayeva et al. 1984a, b), in the other series EH was held at zero potential during the treatment. The time of the treatment in both series was fixed at 10 rain.

Average values of changes in gating parameters after the WSC treatment both at normal and at zero potential are shown in Table 1. Both carbodiimides used induced approximately the same alterations in channel gating, results with EDC and CMC were therefore pooled together when averaging.

Steady-state parameters of sodium conductance in normal membrane

The description of np(E) and h~o(E) curves by the empirical Eqs. (1) and (3) gave the following parameters of these

33

Table 1. Effects water-soluble carbodiimide (100 mM, pH 4.9-5.1, 10 rain) on parameters of sodium channel gating. Data presented as mean _+ SEM (number of experiments)

WSC at holding potential from - 80 to - 100 mV

WSC at zero holding potential

Statistical significance of the difference between effects of two types of the treatment (t-test)

Activation Z e f / Z e f a 1.66 _ 0.07 (t0) 1.02 _ 0.02 (12) **** ag/ag a 1.49 _+ 0.08 (t0) t.29 _+ 0.08 (8) * AEg (mV) 12.5 + t.2 (10) ].1 • 1.8 (8) **** vp"/% (--40 mV) 1.92_+0.18 (10) 1.57_+ 0.08 (9) * ~v"/% (0 mV) 2.94_+ 0.30 (10) 1.56-+ 0.12 (9) ****

Inactivation a~/a~ a 1.95 -+ 0.16 (10) 1.44 • 0.05 (8) *** AE~(mV) 1.1 _+3.6 (10) --9.8 _+3.0 (8) ** ~hfa/%f (0 mY) 4.3 _+ 0.9 (5) 1.6 _+ 0.3 (3) ** �9 h~"/~(0mV) 4.2 --+1.1 (5) 1.7 -+0.3 (3) *

" Parameter relevant to treated membrane * P > 0.05; ** P < 0.05; *** P < 0.01; **** P < 0.001

dependencies: steepness factors of activation and inactiva- tion curves were on average 0.132 _+ 0.004 m V - t (30) and 0.122 + 0.002 m V - 1 (29), respectively, and the potentials of half-activation and half-inactivation were - 25.3 • 2.4 mV (30) and - 77.9 • 2.0 mV (29), respectively. These values are equal, within experimental error, to the corresponding parameter values obtained earlier (Mozhayeva et al. 1980).

The limiting logarithmic slope of the np(E) curve in normal membrane varied considerably from fibre to fibre; the corresponding values o f effective charge o f activation ranged from 3.5 to 5.8 electronic charges (e. ch.). On average, Zof is equal to 4.51 • 0.11 e.ch. (30). This value is a little less than the analogous value obtained in previous studies on the nodal membrane (Chiu 1980; Mozhayeva et al. 1984a, b) and very close to that in squid axon (Oxford 1981).

Effect of the WSC at normal EH

The results o f this series o f experiments are in good qualitative and quantitative agreement with the findings obtained in the previous work (Mozhayeva et al. 1984a, b).

Apparent time constant of activation increased by factors o f about 2 and 3 at - 40 and 0 mV, respectively. The middle part of the nv(E ) curve became 1.5 times less steep and shifted to the right. On average, a 10 rain treatment resulted in a decrease of effective charge of activation by a factor o f 1.66. In the previous work (Mozhayeva et al. 1984a, b) where the treatment time in each experiment was long enough to attain maximal changes in gating properties the corresponding figure was 1.78. Thus a 10 rain treatment produced an effect which was only slightly weaker than the maximal one.

The same alterations in the rate of current rise and in the voltage dependence of np were induced by the WSC in the membrane pretreated with alkaloids aconitine or 7,8- dihydrobatrachotoxinin A, an analog ofbatrachotoxin. This observation gives additional support to the notion that in- activation, which is differently pronounced in normal and alkaloid-modified channels (see Khodorov 1985), does not influence significantly either the time course o f the current rise or the voltage dependence of channel openess, at least in nodal membrane.

Properties of inactivation also changed after the WSC treatment. The steepness o f the h~(E) curve decreased almost two-fold. On average, the half-inactivation potential remained nearly unchanged after the treatment. At potentials more positive than - 40 mV h~ in treated mem- brane did not fall as a rule lower than 0.1, and at further depolarization the h~(E) curve tended to rise (see Fig.2). Non zero level of h~ is reflected in a significant steady-state current component, which in most cases was larger than that in the untreated membrane. Due to a higher level o f h| at E more positive than - 50 mV currents measured with depolarizing prepulses in treated membrane were frequently larger than analogous currents in the unaffected membrane despite the inhibition of maximal sodium conductance.

The two-exponential pattern of current inactivation re- mains after the treatment with both time constants being increased.

After 10 rain WSC treatment at normal holding potential total sodium conductance ranged from 5% to 25% of the initial value.

Effect of the WSC at zero potential

In these experiments the WSC was applied in about 10 s after E . had been switched to 0 inV. After a 10 rain treatment the WSC solution was replaced by the control one and normal E~ level was restored. Prolonged depolarization results in so-called slow inactivation of sodium channels (Fox 1976). Therefore currents measurements following WSC treatment at zero potential several minutes after switching En to the normal level to allow the channels to recover from the state of slow inactivation.

The results of two experiments with application of the WSC at zero potential are shown in Figs. 1 and 2.

The most striking difference of WSC treatment at zero potential from that at normal potential was that it resulted in essentially no change in the Zef value. Thus in the experiment presented in Fig. 1, Zef decreased by no more than 5%. In the rest o f analogous experiments this decrease was still less and was on average 2% (see Table 1).

In three experiments the WSC was applied twice: first at zero and then at normal potential. Figure 2 shows one of

1.0-

O5

o a

34

np 0,I~

0.01-

J ' 1 ' ' ' ,~ .~~ . . . . I ,~ //11 , r ,

-150 -100 -50 0 mV

- 60 -50 -40 mV

.% ms

~ -0,4

2 "0,3

-0,2

-0,1

, i t , , i ~ - 0

-SO 0 mV F i g . 1 a--e. Effect ofWSC (EDC) treatment at zero holding potential on activation and inactivation of sodium conductance. All measure- ments were carried out in control solution at E . - 90 mV. Curves 1 and 2 are relevant to sodium conducatance in normal and treated membrane, respectively, a Voltage dependence of activation (rtp) and inactivation (h o0). b Semilogarithmic plot of the np versus membrane potential. Only lower part of np dependence (np < 0.1) is shown. Corresponding Zof values are 5.74 and 5.48 e.ch. before and after the treatment, respectively, c Voltage dependence of apparent time constant of activation

3 np

0,5~

0

a -1oo o So mV

% m s

np ~ 2 0.5

3 -0.3

-0.2

001 -0.1

b ' - 5 rO ' - 4b ' - 30 rn~ / c l , , i , l i - 0

- 50 0 mV

Fig. 2 a--c. Effects of' two successive applications of WSC (EDC) to the nodal membrane, first at E. 0 mV and then at EH -- 100 mV. Curves 1, 2 and 3 correspond to control, after the first and after the second treatments, respectively, a Activation (np) and inactivation (h~) as functions of E. b Voltage dependence of activation over the voltage range where np< 0.1, semilogarithmic plot. Corresponding Zef values are 3.90, 3.87 and 2.18 e.ch. for normal membrane and after the first and second application of WSC, respectively, c Appar- ent time constant of activation as function of E

such experiments. I t can be seen that WSC at zero En did not alter the limiting logari thmic alope of the %(E) curve, whereas successive t reatment at - 90 mV resulted in a 1.78- fold decrease of this slope and, consequently, Zof. F ina l al terat ions of gating parameters in these experiments were essentially the same as those in the experiments where WSC was applied only at normal potential .

I t can be seen from Figs. 1 and 2 and f rom Table 1 that the rest of gating parameters are altered after the t rea tment at zero potent ia l but a lesser degree and in somewhat dif- ferent fashion than after the t reatment at high negative po- tential.

Kinetics of current rise becomes slower over the entire potent ia l range tested. This slowing, however, is not so strong as after the W S C t rea tment at normal potential . The difference between the two types of the t reatment is especially prominent for currents at large depolar izat ions; thus at 0 mV % increased on average by the factors 1.56 and 2.94 after the WSC at 0 mV and - 80 to - 100 mV, respectively.

The potent ia l o f half-act ivat ion shifted after WSC at 0 mV sometimes of the left (Fig. 1), sometimes to the right (Fig. 2) and, on average, remained at the same value, whereas WSC at normal potent ia l p roduced invariable shift of Eg toward more positive potentials with the mean value of 12.5 inV. WSC applied at zero potent ia l induced a decrease in the steepness of the middle par t of the act ivat ion curve

on average by the factor of 1.29. This value is lower than the corresponding value (1.49) obta ined from measurements with the WSC treatment at normal potentials. However, more experiments are required to establish convincingly whether two types of the WSC treatment reduce this parame- ter differently or not. The bo t t om of the %(E) curve (% < 0.1) after WSC treatment at 0 mV was shifted to the left wi thout a change of the logari thmic slope (see Figs. 1 and 2) whereas after the t reatment at high negative potentials this par t of the curve was si tuated to the right of the control curve and intercepted it if measurements extended to sufficiently negative potentials.

The WSC treatment at 0 mV resulted consistently in the negative shift of the middle par t of the hot(E) curve; change in Eh averaged - 9 . 8 mV whereas after the t reatment at normal holding potentials Eh on average remained in the same posi t ion (see Table 1). The WSC induced a decrease in steepness of the middle par t of the h~(E) curve being applied both at zero and at normal potentials, but its appl icat ion at zero potent ia l decreased a~ less than analogous appl icat ion at high negative potentials. WSC induced on average decreases of ah by factors 1.44 and 1.95 in the first and second cases, respectively. Judging from mean values of the ratios of inact ivat ion constants in the treated membrane compared to those in the normal one (Table 1), we conclude that membrane depolar iza t ion weakens the effect of WSC on the rate of inactivation. The difference between effects of

35

WSC at normal and zero potentials on the fast time constant of inactivation at 0 mV is statistically significant, in spite of the small number of experiments. More experiments are required to determine the behavior of the slow time constant.

The inhibition of sodium conductance after the WSC treatment at 0 mV was approximately the same as after the treatment at normal En.

Control experiments

Two series of experiments were performed with the aim of examining aftereffects of treatment conditions (low pH, zero holding potential) on the channel gating. None of these deviations from the normal conditions altered the limiting logarithmic slope of activation, steepness of the middle parts of the both activation and inactivation curves. External perfusion of the membrane with a solution of low pH at zero potential resulted in shifts in the middle parts of the rip(E) and h~(E) curves toward more negative potentials by about - 3 and - 7 mV, respectively. The time constants of activation over the voltage range from - 4 0 to 0 mV in- creased by 1 0 - 2 0 % . After the analogous perfusion of the membrane with acid solution at normal holding potential changes in the gating parameters were in the same direction but smaller. Some minor part of the observed changes in gating after the WSC treatment could thus be ascribed to low pH and prolonged depolarization. The comparison of these ,,nonspecific" effects with the final effects of the WSC treatments suggests that the contribution of "nonspecific" components to overall changes is larger when WSC is applied at zero potential. Thus after correction for the "nonspecific" components the difference between "specific" effects of WSC at normal and zero potential would be even more pronounced.

Discussion

The results given here are in qualitative agreement with the hypothesis of mobile carboxyl groups (Mozhayeva et al. 1984a, b). According to this hypothesis, part of mobile gating charges are presented by COO- groups which are located at the external surface of the channel molecule when the channel is closed and move into the membrane as the channel opens or gets inactivated. Modification of these groups with the WSC either changes their charge and/or immobilizes these groups. It should lead to a decrease in the amount of displaced charge associated with channel opening, i.e. in the decrease of Zof. At zero potential when channels are open or inactivated, mobile groups are expected to be in the depth of the channel structure and, consequently, inaccessible to the WSC. The fact that prolonged depolariza- tion, when channels are inactivated and not opened, prevents the WSC-induced decrease of effective activation charge suggests simply that charges displaced into the membrane as the channel opens do not return back to the surface when the channel passes into states of fast and then slow inactivation.

The effective charge carried by each mobile group during gating is equal to the group charge multiplied by a fractional distance it travels in the electric field. After the WSC treat- ment Zof decreased in some experiments by up to three e. ch., hence the number of modified mobile groups is at least three. Since the distance travelled by each particle is likely to be

outside

Fig.3. Mechanical representation of mobile (O) and fixed ([]) charges in gating mechanism of sodium channel. Position of the mobile charges on the membrane surface corresponds to closed state of the channel, position in the depth of the channel structure corresponds to open or inactivated states. See text for explanation

rather short, the number of reacted particles would seem to be much more than three.

Modified channels continue to be electrosensitive, which suggests they possesss mobile particles. It is likely that these remaining charges are presented by COO- groups which are either inaccessible to the WSC or modifed in such a way that channels cannot open and therefore become invisible to our measurements. It is possible that such kinds of modifications are responsible (along with other mech- anisms) for the WSC-induced conductance inhibition.

Channel opening is a multistep process (Hodgkin and Huxley 1952; Gilly and Armstrong 1982) and the total Zof is a sum of charges associated with each step from the "fully" closed to the open state. Thus, the Z~f decrease can be the result either of a decrease in the charge of each step and/ or a reduction of the number of steps leading to channel opening.

The fact that the WSC treatment at zero potential in- duces some alterations in gating functions makes it necessary to assume additionally the existence of carboxyl groups which are of some importance for operation of the gating machinery, and, unlike mobile groups, are accessible for WSC at any membrane potential. These groups can be called permanent surface groups or fixed charges. The existence of negatively charged groups fixed on the membrane surface has been postulated also in the so-called surface potential model (Frankenhaeuser and Hodgkin 1957). This model predicts that neutralization (or screening) of these groups, for example, due to enhancement of Ca 2 + ion concentration, should result in a parallel shift of the %(E) and h ~(E) curves along the voltage axis toward more positive potentials. As COO- groups lose their negative charge after reaction with WSC, the effects on channel gating according to this model might by expected to be qualitatively analogous to those of Ca 2 + or H + ions, with the only difference that the WSC effects are irreversible. Contrary to this expection, the WSC treatment at zero potential induces no or even negative shifts (see Results). Hence the modification of permanent surface acid groups, if it takes place, affects channel gating in ways different from those suggested by the surface charge model. Firstly, it may be assumed that after the modification of permanent surface groups some secondary conformational rearrangements occur which are of greater importance for the gating machinery than alteration of the charge itself. Thus, the modification may change relative energetic levels of possible states of the gating machinery which, in turn, may lead to changes in kinetics and potential dependence of gating. I f these changes are such that states intermediate between "fully" closed and open grow more stable than in

36

normal channels, the steepness of the middle par t of the %(E) curve (parameter ag) may decrease without changing the effective charge of activation.

Secondly, due to the discrete nature of the charges and also to the assumed abil i ty o f the mobile charges to emerge at the surface, even pure electrostatic effects of fixed charges on the gating may not be as unequivocal as the surface model (at least in its original form) predicts. This poin t may be explained with the help of Fig. 3, where two fixed and two mobile charges are depicted. Coulombic repulsion between groups of the left pai r is s tronger when the mobile charge in the membrane is in the "closed" posi t ion (i. e. towards the membrane outside surface). Thus, the presence of negative fixed charge will favour the channel to be in the open (or inactivated) state. I f this fixed group is neutralized, the p rob- abili ty for channels to be open decreases at given potential , which will be reflected in the shift of the np(E) curve to the right, in full agreement with the surface charge model. However, charges may be ar ranged in such a way that cou- lombic repulsion between the mobile and fixed charges will be stronger when the mobile charge is in the "open" posi t ion (right pair). I t is clear that in that case the effect of the fixed charge on the dis t r ibut ion of the mobile one between the "closed" and "open" (inactivated) states will be entirely op- posite to that suggested by the surface charge model.

Thus possible consequences of the modif icat ion of assumed surface C O O - groups may be very different and not predictable beforehand with the da ta available. The variabi l i ty of some WSC-induced al terat ions in gating parameters , for example of the np(E) curve shift after the WSC treatment at zero potential , is likely to be due to the variabi l i ty of differently directed individual al terations.

The proposa l that C O O - groups play a role of mobile gating charges is at tractive but one should keep in mind that al ternative explanat ions o f the da ta are possible. Thus the da ta can be explained on the assumpt ion that modif iable C O O - groups are not mobile themselves but are in some contact with mobile structures. Then their modif icat ion with WSC would indirectly cause restriction in mot ion of gating particles and thereby in the Z~y decrease. The weakening of the effects o f WSC at zero potent ia l could then be explained, for example, by the assumption that mobile structures in open or inactivated conformat ions screen C O O - groups from WSC attack.

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Received March 9/Accepted September 5, 1985