Cambridge CB2 3EG (Received 6 March 1970)

Transcription

1 J. Phygiol. (1970), 209, pp With 10 text-ftgures Printed in Great Britain THE EFFECT OF THE TETRAETHYLAMMONIUM ION ON THE DELAYED CURRENTS OF FROG SKELETAL MUSCLE BY P. R. STANFIELD From the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG (Received 6 March 1970) SUMMARY 1. A method which permitted control of the membrane potential near the end of a muscle fibre and measurement of an approximation of the membrane current was used to investigate the effects of the tetraethylammonium (TEA) ion on the delayed outward potassium current obtained on depolarizing. 2. Assuming Ri to be 250 Q cm and a fibre diameter of 80,u, the mean value for the maximum potassium conductance (H) was X2 mmho. cm mm-tea, in two series of experiments, reduced gk by about 90 %. A concentration-effect relation for TEA in its action on the delayed rectifier could be fitted by a curve for a drug-receptor complex assuming one molecule of TEA to combine reversibly with one receptor, and a dissociation constant of 8 x 103 M. 4. TEA tended to shift the threshold for delayed rectification to slightly more negative membrane potentials. TEA caused a similar shift in the relation between noo and membrane potential, but did not much alter the form of the relation. 5. The relation between n'0 and membrane potential and between r.-1 and membrane potential were well fitted by the model of Adrian, Chandler & Hodgkin (1970a) assuming that the Q10 was TEA slowed the rate of onset of the delayed potassium currents, decreasing Tn-1 (the reciprocal of the time constant of the fourth power function which described the current's development) by about 80 %. 7. The inactivation of the delayed current with time was shown to follow a complex time course. A fast phase decays with a time constant of 270 msec and a slow phase with a time constant of 2-3 see at a membrane potential of + 10 mv. 8. The fast phase of the delayed current is much more susceptible to the

2 210 P. B. STANFIELD action of TEA than the slow phase, and these are interpreted in terms of different potassium channels. TEA has little effect on the time constant with which either the fast current or the slow current inactivates. INTRODUCTION The aim of the experiments described in the present paper and in the following one (Stanfield, 1970) was to investigate the actions of the tetraethylammonium (TEA) ion on the potassium permeability of frog skeletal muscle. TEA is an important agent in physiological experiments. Aside from its curariform actions (Raventos, 1937), it has the effect in many nerve preparations of reducing the time dependent changes in potassium conductance which occur when the surface membrane is depolarized (e.g. squid axon: Tasaki & Hagiwara, 1957; Armstrong & Binstock, 1965; node of Ranvier preparations from amphibia: Schmidt & Stimpfii, 1966; Hille, 1967; Koppenh6fer, 1967; sub- and supra-oesophageal ganglion cells of a pulmonate mollusc Onchidium: Hagiwara & Saito, 1959; supramedullary cells of the puffer-fish Spheroides: Nakajima, 1966). Hille's results (Hille, 1967) suggest that, at the frog node of Ranvier, TEA may well be as specific in its actions on the delayed rectifier (the channel which carries the time-dependent potassium current) as tetrodotoxin (TTX) is on the sodium channel (for references, see Kao, 1966). Thus TEA was shown to affect only the potassium conductance, 9K, and to be without effect on the sodium conductance, gna, leak conductance, gl, or on the kinetics of the sodium or potassium conductances. While the results of Schmidt & Stimpfli (1966) and Koppenhbfer (1967) are not in complete agreement with those of Hille (1967), TEA being found to alter rates of sodium inactivation and potassium activation, the position in nerve is a comparatively straightforward one. The ionic permeability of skeletal muscle is rather more complex, however, and this is reflected in the greater complexity of TEA's actions (see also Stanfield, 1970). TEA is known to prolong the action potential of frog skeletal muscle (Hagiwara & Watanabe, 1955; Washio & Mashima, 1963) and has been shown to reduce the amplitude of delayed potassium currents in this preparation (Kao & Stanfield, 1970). The present experiments examine in more detail TEA's action on the delayed potassium current, analysing the records in terms of the model of Hodgkin & Huxley (1952). The experiments also demonstrate the relative resistance to TEA of a second more slowly developing outward current obtained on depolarizing.

3 TEA AND MUSCLE DELA YED RECTIFICATION 211 METHODS Voltage clamp. The voltage clamp technique used to obtain the results of this paper and of the following one (Stanfield, 1970) was developed by Adrian, Chandler & Hodgkin (1966). It involves the impalement of a muscle fibre near its end with three micro-electrodes (Fig. 1). The two recording electrodes (recording V1 and V2) impaled the fibre at distance 1 and 21 respectively from the fibre end and were filled with potassium chloride; they had tip resistances usually of 7-12 Mf2 and tip potentials of less than 5 mv. The current electrode was filled with 2 M potassium citrate and had a tip resistance of 3-5 MQ. This electrode was screened to within 2-3 mm of its tip. The potential difference V1 between the inside of the fibre and the bathing fluid was recorded on one beam of a Tektronix 502A oscilloscope and was also led, through cathode followers, to the vertical amplifier of a second oscilloscope which served as a feed-back amplifier. The gain of this was generally set at 10,000, but sometimes had to be reduced to 4000 to avoid oscillations in the feed-back loop. A calibrator, C, was used to determine the holding potential, and square pulses were fed in at S using a Tektronix type 161 pulse generator, together with decade potentiometers. Adrian etal. (1970a) show that the membrane current is given by the approximation Im =a(v2l VDA Z (1) where a is the fibre radius, V2 and V1 the membrane potentials at x = 21 and x = I respectively (see Fig. 1), and Ri is the specific internal resistance of the fibre. The membrane current was thus recorded as V2 - V1 through cathode followers whose differentiality was adjusted as exactly as possible. The total electrode current (I.) was recorded as the IR drop across a 2-2 kq resistor, which was shunted by a capacitor giving this circuit a time constant of 0*6 msec. Adrian et al. (1970a) show that the approximation for the membrane current is correct to within 5% if I is less than 2A and that it follows from this that (V2 -VIV V must be less than 6. Other errors, due to imperfect differentiality between the two recording electrodes and their cathode followers, become large if I is made too small. To avoid errors from both sources, I was varied between 125 and 250 /s; and in no case was the condition (V2 - V1)/VI < 6 exceeded. Experiments were performed in two series. In the first, which investigated the effects of several concentrations of TEA, I was set at 200 #a throughout, In the second series, I was set at 125 pa for experiments with hypertonic standard Ringer and at 250,# for experiments with 58 mm-tea chloride Ringer. The sartorius muscle of the frog (Rana temporaria) was used throughout the present experiments. Solutions. The hypertonic standard Ringer solution used in the experiments of this paper was identical in ionic composition and ph with that used by Adrian (1956) but contained additional sucrose at a concentration of 350 mm. This increased the tonicity of the Ringer to about 2 j times normal and very much reduced the ability of the muscle to contract (Howarth, 1958), TEA Ringer solutions contained various concentrations of TEA chloride (Table 1) which replaced sodium chloride on a mole for mole basis. All solutions contained TTX at a concentration of 10-7 g/ml. Experiments were performed at room temperature, 20-23o C. Student's t test was used for comparisons for significance of difference.

4 212 P. R. STANFIELD I I x=21 x=i x=o Fig. 1. Diagram illustrating circuit. The feed-back amplifier, used at gains of 4000 or 10,000, is represented by FB and the membrane potential is recorded at V1. Membrane current is recorded at (V2 - V1) and the total electrode current at Io as the IR drop across a 2-2 kfl resistor. C represents a calibrator and S the square-wave source. The two recording electrodes, recording V1 and V2, impaled the fibre at distances 1 and 21 respectively from the fibre end (x = 0). It may be shown that membrane current IM = a(v2-vi) where a is the fibre radius and R, the internal resistivity of the fibre. RESULTS Voltage-current relations in hypertonic standard and TEA Ringer. Figs. 2 and 3 show typical records and voltage-current relations from two fibres: one in hypertonic standard and the other in hypertonic 58 mm-tea chloride Ringer. The results are from the second series of experiments where the fibres were clamped at a holding potential of mv, those from the first series being entirely comparable. It is clear from the two Figures that TEA reduces the amplitude of the delayed current. (It should be noted that the inter-electrode distance, 1, is twice as long in the case of the fibre in TEA as in that in hypertonic standard Ringer, and this effectively increases the gain fourfold.) In the second series of experiments, the holding potential was mv, and in the first series -90 mv, for all fibres whether in hypertonic standard or TEA Ringer. But it was found that the resting potentials were lower (less negative), in TEA chloride solutions made hypertonic with 350 mm sucrose. The effect may probably

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6 214 P. B. STANFIELD be attributed to TEA's action in reducing the resting potassium conductance (Stanfield, 1969, 1970) which results in the resting potential being more dependent on sodium and on chloride, if this is not in equilibrium. The values for the resting potential obtained in various concentrations of TEA (in hypertonic Ringer) are given in the second column of Table 1. The depolarization in 58 mm-tea chloride, for example, is about 7 mv and is statistically significant (P < 0.001). Reduction of delayed currents by TEA. Subtraction of leak current shows that the effect of TEA is to reduce the amplitude of the potassium current, A Hypertonic standard l=125/i :H11 I -'. -- D D20 D30 t D >n L 8 58 mmtea chloride 1=250,a DI20 k D 31 - let ~D41 c - D51 Q61 ~~!~- D71 D61 D71 EI - LA - 2 H 20 msec D7A V I - E I rd -!I )85 H 20 msec Fig. 2. Records of membrane current (V2-V1) obtained from two fibres during square hyperpolarizing and depolarizing voltage pulses. A, fibre in the hypertonic standard Ringer; RP: -97 mv; HP: mv; T0: C; inter-electrode distance, 1: 125 /z. B, fibre in hypertonic 58 nm-tea chloride Ringer; RP: -83 mv; HP: -100 mv; T0: 21-2' C; 1: 250 4u. Fibres are the same as those of Fig. 3. Numbers at the right of each trace give the amplitude of the hyperpolarizing (H) or depolarizing (D) pulse in mv from the holding potential (- 100 mv). Thus the membrane potential during pulse D 10 would be -90 mv. D83 D95

7 TEA AND MUSCLE DELA YED RECTIFICATION 215 and hence, if the equilibrium potential is not altered by TEA, of the potassium conductance. In hypertonic standard Ringer the equilibrium potential was measured in a few fibres and was between -70 and -80 mv (comparable to the values of Adrian, Chandler & Hodgkin, 1968). In TEA, it was unfortunately not possible to measure with any accuracy the equilibrium potential because the tails occurring when the depolarizing pulse was switched off were so small. However, it was clear that the equilibrium potential was not greatly affected by TEA. The smallness of the Ss 50 A Hypertonic standard Ringer EE!2S I >8 B 58 mm TEA Ringer _ -80 _ D o _5 VI (mv) -2 VI (my) Fig. 3. Voltage-membrane current relations from fibres in A, hypertonic standard Ringer, and B, hypertonic 58 mm-tea chloride Ringer. Abscissae: membrane potential (V1) in mv; ordinates: peak currents plotted as V2-V1 (my). Note that the scales are different in the two parts of the Figure. The fibres are the same as those of Fig. 2. inward current tails obtained when the membrane potential was returned to a value negative to the equilibrium potential may be taken as evidence that TEA blocks inward as well as outward currents. It is clear from the results of Figs. 2 and 3, then, that TEA reduces the conductance change which underlies delayed rectification. From voltagecurrent relations of the kind shown in Fig. 3A, it is possible to obtain an approximate value for 9 (the maximum potassium conductance). To do this delayed currents had to be converted to conductances, making certain assumptions: first, that the equilibrium potential of the delayed rectifier was -80 mv (Adrian et al. 1968); secondly, that the diameter of the muscle fibres used was 80 /a; and thirdly, that the internal resistivity of the fibres was 250 cm. The second assumption seemed reasonable from approximate measurements of the diameter of superficial fibres in the whole sartorius in hypertonic Ringer with a micrometer eye-piece and 100 x magnification,

8 216 P. R. STANFIELD and the third assumption from measurements by S. Nakajima (unpublished observations) on single fibres. The fibre of Figs. 2A and 3A thus had a E of 28 mmho. cm-2 compared with a value for ge of 27.0 mmho. cm-2 at 0 mv, 22 5 mmho.cm-2 at -10 mv and 14 8 mmho. cm-2 at -20 mv. The mean value for ge from both series of experiments in hypertonic standard Ringer is *2 mmho. cm-2 (ten fibres). If Hille's figure of 109 QL for the resistance of a single ionic channel (Hille, 1968) applies here, there will be of the order of 2*5 x 107 potassium channels per cm2 of membrane surface. It may be noted that the potassium conductance is similar to that found in the squid axon at 6 0 by Hodgkin & Huxley (1952). The fibre of Figs. 2B and 3B had a ge of 1x7 mmho. cm-2, with a mean value from both series of experiments in hypertonic 58 mm-tea chloride Ringer, of mmho. cm-2 (fifteen fibres). TEA chloride at a concentration of 58 mm thus reduced the delayed conductance by about 90 %. The percentage reductions in E found in the presence of other concentrations of TEA are given in column 3 of Table 1. It must be pointed out that the values obtained for gk in the experiments of series I were consistently lower than those in series II. Thus in hypertonic standard Ringer, g. was 15-1 ± 1 8 mnho. cm-2 (five fibres) in the experiments of the first series and mmho. cm-2 (five) in the second series. Similarly, g9 was mmho. cm-2 (eight) in the first series of experiments in 58 mm-tea chloride and nmnho. cm-2 (seven) in the second. The reason for this difference is not clear. It is unlikely to be due to differences in holding potential since there is no steady-state inactivation of the potassium conductance at -90 mv (Adrian et al. 1970a; see also Kao, & Stanfield, 1968) but might be due to some seasonal variation, the experiments of the first series being performed during winter months (Nov.-Jan.) and the second during the following April-June. Quaternary ammonium ion receptor. Fig. 4 shows a concentrationeffect relation for the actions of TEA on the delayed rectifier. The experimental points fit, fairly closely, the curve for a drug-receptor complex with a dissociation constant of 8 x 10-3 M, assuming that one molecule of drug combines reversibly with one receptor. The fit of the experimental points to the theoretical curve is not perfect, but is apparently better than that to a curve drawn assuming two molecules of TEA need to combine to a single receptor, a curve which is also shown in Fig. 4. The dissociation constant has a value 20 times that found by Hille (1967) in the frog node of Ranvier, similar reductions in delayed conductance to those found by Hile being obtained in Xenopu8 node by Koppenh6fer (1967). It may also be pointed out that the present results, obtained with the voltage clamp, compare reasonably well with the concentration-effect relations found by Hagiwara & Wantanabe (1955) and Washio & Mashima (1963) using action potential duration.

9 TEA AND MUSCLE DELA YED RECTIFICATION 217 One of the assumptions made in computing dose-response curves such as those of Fig. 4 is that the effect of the agent is proportional to the number of receptor sites filled. One explanation of this kind of assumption in the present case is that each ionic channel has a single receptor site, so that the channel might be expected either to be blocked or to be quite unaffected by the agent. If the channels which were not blocked by TEA were completely unaffected by it, TEA should have no effect on the rate constants of the Hodgkin-Huxley model (Hodgkin & Huxley, 1952) which determine the way in which the potassium current turns on on depolarization. It appears that this may not be the case in muscle '.~"" g~~~~~~~~~~ *%s 40 %~~S 20 \.'*- _ 0.* ' U U MI'A TEA concentration (mm) Fig. 4. Concentration-effect relation for the action of TEA on the delayed rectifier. Abscissa: TEA concentration in mm (log scale); ordinate: maximum potassium conductance, 9K, as a percentage of that in hypertonic standard Ringer. During the first series of experiments (0) the holding potential was -90 mv and during the second series (0) mv. The points give means from two to ten fibres. Dashed and dotted line: theoretical curve for drug-receptor complex, assuming one molecule of TEA combines reversibly with one receptor and a dissociation constant of 8 x 10-3 M; dashed line: assuming two molecules of TEA combine reversibly with one receptor. Effect of TEA on n, and the threshold for delayed rectification. Kao & Stanfield (1968) examined the effects of the lyotropic anions on the mechanical threshold and on the threshold for delayed rectification. Using a similar criterion to obtain the threshold for delayed rectification, it was found, here, that TEA tended to shift the threshold to slightly more negative membrane potentials, though only in 29 mm TEA chloride is the shift significant (P = 0 02). The results are given in column 4 of Table 1. Similar effects were seen by Costantin (1968) and Kao & Stanfield (1970). Analysis of the results in terms of the equations of Hodgkin & Huxley (1952) show that the relationship between no, and membrane potential is also shifted along the voltage axis in TEA although the form of the relationship is little altered (Table 1, column 5; Fig. 5).

10 218 P. B. STANFIELD It is not considered necessary to discuss in any detail the model used to describe the potassium conductance changes which underlie delayed rectification in muscle: this is identical in most respects with that of Hodgkin & Huxley (1952) for conductance changes in the squid axon, as Adrian et al. (1970 a) have shown. Thus, although the delayed conductance inactivates with time in muscle (Adrian et al. 1966; see also below Fig. 8), this inactivation is slow enough to be ignored in an analysis of the turnon of the conductance i El~.' _ ~~~~~~~0-2 i 0* Membrane potential (my) Fig. 5. Relation between no and membrane potential. Abscissa: membrane potential (mv); ordinate: na,. The values for n,0 were determined from the voltage-current relations of the experiments of the second series (see Fig. 3) in the hypertonic standard Ringer (0) and in hypertonic 58 mm TEA chloride Ringer (M). The vertical bars give the S.E. of the mean for the values in the standard Ringer. The dashed and dotted line was determined from the model given by Adrian et al. (1970a), assuming only that a.c and fi. were increased 6-12-fold over their values at 1-3O C, i.e. assuming a Qj0 of (Relations between ac., fi and membrane potential are given in Table 6A of Adrian et al. 1970a; other constants in column 3 of their Table 6B.) It follows from eqns. (6) and (7) of Hodgkin & Huxley (1952) that n. at a given membrane potential is defined as follows: n an (2) where gk< is the peak potassium conductance at that membrane potential, gk is the maximum potassium conductance, and a, and,/? are rate constants which vary with membrane potential but not time.

11 TEA AND MUSCLE DELA YED RECTIFICATION In describing the turn-on of the potassium current in the present experiment, eqn. (11) of Hodgkin & Huxley (1952) may be simplified to where 219 =K = [1 exp (-t/rn)]4, (3) since no has a very small value at the holding potentials used here, and gk may be assumed to be zero at zero time. A suitable way of describing the shifts in the relationship between n, and membrane potential in the presence of TEA chloride is to compare the membrane potentials in hypertonic standard and TEA chloride Ringer at which n.0 = 0-5 (i.e. where ot. = fin from eqn. (2) above). This value of the membrane potential is called Vin in column 5 of Table 1, where the experimental results are given. In the experiments of series I, significant shifts in Vin were found in 29, 58 and 115 mm TEA chloride (P values 0-003, and respectively). In the experiments of series II, the 2-3 mv change in Vin in 58 mm-tea is not significant (P > 0.33) but is in the same direction as that in the experiments of the first series. The shift in the relationship between n<, and membrane potential is also in the same direction as that found for the threshold for delayed rectification, and presumably underlies it, although the size of the shift is not always the same in the two cases. It may be pointed out that n:,, generally has a value between 0-2 and 0-3 at the threshold for delayed rectification. The mechanism whereby TEA shifts the threshold for delayed rectification and the relationship between ne* and membrane potential is unclear. However, it may well be similar to that whereby another quaternary ammonium ion, choline, alters the relation between tension and membrane potential (Hodgkin & Horowicz, 1960a), especially since Costantin (1968) and Kao, & Stanfield (1970) have found that shifts in the presence of TEA of the threshold for delayed rectification were accompanied by parallel shifts in mechanical threshold. Hodgkin & Horowicz (1960a) suggested that the important factor in this action of choline was the replacement of extracellular sodium and that the mechanism was similar to the sodiumcalcium antagonism postulated by Luttgau & Niedergerke (1958) for their observations on cardiac muscle. If such an explanation can be applied here for the effect of TEA on n,, this effect cannot be considered as evidence against the view that the form of the dose-response relation for the action of TEA on g. can be explained in terms of one receptor for one ionic channel. Effect of TEA on Tn. Fig. 6 shows potassium currents (the currents of Fig. 2A and B after subtraction of leak and capacity currents) obtained with the voltage clamp and fitted by eqn (3). (In doing this, currents were

12 220 P. R. STANFIELD worked in terms of V2 - V1, which is proportional to conductance). The experimental results are fitted well by the fourth power function, just as in the case of the squid axon (Hodgkin & Huxley, 1952). Fig. 7 gives the 40 A Hypertonic standard Ringer 30 v C msec 15 ) msec Fig. 6. Delayed currents fitted by the Hodgkin-Huxley model (Hodgkin & Huxley, 1952). Abscissae: time (msec); ordinates: symbols, IK in terms of V2 - V1 less leak and capacity currents; lines, 'K determined from the equation 'K = 1K. [1-exp (-t/rt)]4. Numbers at the end of each trace give the amplitude in mv of the depolarization from the holding potential of mv. Photographic records of these currents, without leak or capacity currents subtracted, are given in Fig. 3. Thus fibre A is that of Figs. 2A and 3A and fibre B that of Figs. 2B and 3B.

13 TEA AND MUSCLE DELA YED RECTIFICATION 221 A Hypertonic standard Ringer an 2 0~~~ (.0 B 58 mm TEA Ringer 0@3 0 Cb*0 AL~~~ Aa T LA e 0 2 WV#$tf+ * V 0-1 I I I I I I Membrane potential (my) Fig. 7. Relation between 'r-1 and membrane potential, A in the hypertonic standard Ringer and B in the hypertonic 58 mm-tea chloride Ringer. Abscissae: membrane potential (mv); ordinates: reciprocal of Tn (msec-1). Symbols: values for T-71 from five fibres in hypertonic standard Ringer and seven fibres in hypertonic 58 mm-tea chloride Ringer. All values were determined from the turn-on of the potassium conductance except the two left-hand values in A ((I, V) which were determined from the turn-off of potassium conductance using two pulse experiments; these values are from the fibres represented otherwise by 0 and A respectively. The lines fitted to the experimental results were determined from the model given by Adrian et al. (1970a) assuming only that a. and fil were increased 6-12-fold over their values at 1-3 C, i.e. assuming a Q10 of (Relations between a., fib and membrane potential are given in Table 6A of Adrian et al. 1970a; other constants in column 3 of their Table 6B.) Continuous line: r-1 (= a,,+,fi); dashed and dotted line: a.; dotted line /lb

14 222 P. R. STANFIELD values for 1/rn from the fibres of series II in the presence of hypertonic standard and 58 mm-tea chloride Ringer, plotted against membrane potential. It is clear that Tn is larger in the presence of TEA. It is useful to compare the values for Tn found here in the hypertonic standard Ringer at a mean temperature of 21-3 TC with those of Adrian et al. (1970a) at a temperature of 1-3 C. Comparing the present results with theirs over the membrane potential range 0 to -25 mv, where in both cases Tn was determined from the turn-on of the potassium conductance, the Q10 is found to be 2-5. (Tn is 6 1 times larger in their experiments at a temperature 200 C lower.) But at membrane potentials more negative than -25 mv, where Adrian et al. (1970a) have determined Tn from the turn-off of potassium conductance in two-pulse experiments, the apparent difference between the two groups of results is smaller. A possible reason for this is the complication introduced by a second, more slowly developing potassium conductance change found in skeletal muscle on depolarizing (Adrian, Chandler & Hodgkin, 1970b; see also below, p. 225). If the slow conductance change is relatively more important at more negative membrane potentials, the apparent Tn for the turn-on of the delayed conductance will be longer. An argument against this view is that eqn. (3) still fits well the currents found experimentally in this potential range. But two values for Tn from two pulse experiments at -56 and -68 mv (Fig. 7A) are again in reasonable agreement with the results of Adrian et al. (1970a) and a Q10 of 2-5. It may also be mentioned that the relationship between noo and membrane potential found here (Fig. 5) is very similar to that found by Adrian et al. (1970a) and is fitted well by their equations for axn and fn as functions of membrane potential (their Tables 6a and 6b). Also given in Fig. 7A are values for I/Tn, and axn and fin determined from these equations of Adrian et al. (1970a) assuming a Q10 of 2-5. In the presence of 58 mm-tea chloride, 1/Tn was found to be reduced by 82-5 % in the potential range 0 to -25 mv. At membrane potentials more negative than this, the apparent reduction is smaller, presumably, again, because of the complication introduced by the slowly developing potassium conductance. It is also possible that the reduction in 1/Tn in the range 0 to -25 mv might be smaller than is apparent since TEA appears to have a greater effect on the delayed rectifier than it does on the slowly developing conductance (see below, p. 225). However it seems certain that Tn is considerably lengthened since the fit of eqn. (3) to the records made in TEA is good (Fig. 6B) and, as may be seen from the record of Fig. 8Bi, the contribution of the slowly developing conductance to the delayed current is relatively small with large depolarizations in the presence of TEA. It would thus appear that in muscle, unlike nerve (Hille, 1967), TEA

15 TEA AND MUSCLE DELA YED RECTIFICATION 223 has a more complex action on the delayed rectifier than one in which one TEA molecule blocks one ionic channel. It is felt that the present results in the presence of TEA are not adequate for the writing of equations for an and fin of the kind developed by Adrian et al. (1970a). But the present results are not inconsistent with the view that lowering the temperature or the presence of TEA reduces both An and fin, potassium conductance alters with time and membrane potential. A Hypertonic standard B 58 mm TEA chloride E on C20 the potential dependent rate constants which determine the way the _> E~~~ ~ ~ v > -- _4 ', E a E 1-I1 sec 1=125,u i-2sec 1=250u Fig. 8. Records of membrane potential and current during long depolarizing pulses in two fibres. A, in hypertonic standard Ringer; B, in hypertonic 58 mmx-tea chloride Ringer. Record A ii was the twelfth such pulse applied to the fibre with only 3-4 sec between each. Record Bii was the third pulse applied to the fibre in TEA in similarly rapid succession. Note the second hump (vertical arrow) at about 800 msec is relatively resistant to inactivation and to the action of TEA. A, fibre RP: -84mV; HP: -loomv; T0: 20-4 'C. B, fibre RP: -80 mv; HP: -100 mv; T0: C. Both fibres were treated with tetrodotoxin (0.1,ug./ml.). Experiments with long depolarizing pulses. When long depolarizing pulses are applied to muscle fibres immersed in hypertonic standard Ringer (Fig. 8Ai), the delayed current inactivates with an approximately exponential time course. In the record of Fig. 8Ai, where the membrane was depolarized from to + 10 mv, the inactivation had a time constant of 270 msec, this value (270 msec) also being the mean from three fibres. If this time constant is compared with those found by Adrian et al. (1970a) at 1-3 C, the Q10 for this process is about 2-8. But the time course of the inactivation is not a simple exponential, as may be seen when the current of Fig. 8Ai is plotted, after subtraction of the current at the end of the pulse, on a semi-logarithmic scale as in Fig. 9A. A second, slower phase of inactivation is apparent. Further, if several 8 PHY 209

16 224 P. R. STANFIELD successive long pulses are applied to the fibre, the current intensity becomes progressively smaller as the delayed current is not allowed to recover between each pulse. When this happens, a small but definite second 'hump' appears at about 800 msec after the beginning of the pulse (vertical arrow in Fig. 8A ii). In 58 mm-tea, however (Fig. 8B), this second hump is prominent even without inactivating the delayed rectifier (Fig. 8Bi) though it becomes much more prominent when the delayed current is inactivated slightly as in Fig. 8Bii. (The details of this experiment are given in the legend of Fig. 8). 100 A Hypertonic standard so C E 20_ 10\ > A B 58 mmtea C s msec Fig. 9. Semi-log. plot of membrane currents of A, Fig. 8Ai; B, Fig. 8Bi. Abscissae: time in msec; ordinates: membrane current (V2 - Vj) less current at end of pulse (log. scale). Time constants, fast phase: 270 msec (standard), 280 msec (TEA); slow phase: 2-6 sec (standard), 2-5 sec (TEA). Fig. 9 gives semi-logarithmic plots of the currents of Figs. 8A i and 8Bi after subtraction of the current at the ends of the pulses. The two phases of inactivation apparently occur at about the same rate in the presence or absence of TEA, but the fast phase is clearly smaller in amplitude when TEA is present. (It should be noted that the current gain is effectively four times higher in the case of the fibre in TEA because of the difference in inter-electrode distance, 1.) The time constant for the faster phase in the presence of TEA is 290 msec (mean from three fibres) at a membrane

17 TEA AND MUSCLE DELA YED RECTIFICATION 225 potential of + 10 mv, a value very similar to that found in the standard Ringer. The time constant of the slower phase is 2-3 sec (three fibres) in hypertonic standard and 2-1 sec (three fibres) in 58 mm-tea chloride Ringer. The inactivation of the slow phase with time may also be seen from the experiment of Fig. 10. Here the effect of pulse duration on the slow inward tail of current obtained when the membrane potential is returned to 2 '0 58 mm TEA I 01, -I I sec Fig. 10. Records of membrane potential and current (traced) with tails of currents of different durations superimposed. Note that tails become less inward as current decays after second (800 msec) hump on current trace. Fibre RP: -81 mv; HP: -lo1mv; T0: C; TTX: 0 1jug./ml. The fibre was immersed in hypertonic 58 mm TEA chloride Ringer mv has been investigated. The tails become progressively smaller in amplitude when the pulse duration is increased, although pulses of different duration were not applied in any systematic order. Adrian et al. (1970b) concluded that there is a second, slowly developing conductance change to potassium in skeletal muscle. Such a channel would explain the observations described here, made in the presence of standard Ringer, and one can conclude that this channel is relatively resistant to TEA. The way in which this slow current might interfere with the determination oftn has already been discussed. It might also be mentioned here that the presence of this current and its resistance to TEA's action may account for the imperfect fit of the experimental points to the dose-response relation of Fig

18 226 P. R. STANFIELD DISCUSSION The results of the present paper show that TEA acts on skeletal muscle much as it does on nerve to inhibit delayed rectification (Hille, 1967; Koppenhbfer, 1967) but that the affinity of the receptor for the tetraethylammonium ion is considerably lower (by more than one order ofmagnitude) and also that TEA affects the kinetics of the potassium activation. The lower affinity, it seems, is real and not just apparent. It cannot be explained by a very long diffusion delay (perhaps because TEA has to enter the T-system, or because the receptor sites might be on the membrane inner surface) for a number of reasons. First, it is arguable whether there are any delayed rectifier channels in the membrane of the T-system, for passing long depolarizing pulses results in very little change in equilibrium potential for the delayed current (Adrian et al. 1970a). Secondly, the rate of onset of TEA's action on the duration of the active state (which is almost certainly related to the prolongation of the action potential-see Sandow, 1965, for argument for zinc) is rapid, having, in whole muscle, a half-time of about 3 min (Edwards, Ritchie & Wilkie, 1956). It seems likely that the 20 min equilibration time which was used with TEA solutions in the present experiments would be quite adequate. And third, Kao & Stanfield (1970) found that the affinity for the longer chain monoquaternary ammonium ions tetrapropyl- and tetrabutylammonium was higher in skeletal muscle than that found by Hille (1967) with frog nerve. This result would seem quite incompatible with any view that differences in affinities between nerve and muscle are due to quaternary ammonium ions taking a long time to diffuse to their receptor sites. Nor does it seem likely that the receptor sites are on the inner surface of the fibre membrane. Although this point has not been studied directly, it may be noted that TEA is known not to penetrate the squid axon to any great extent (Armstrong & Binstock, 1965) and that other quaternary ammonium ions (TMA, choline) penetrate human red cells but poorly (Askari, 1966). The prolongation of Tn by TEA shown in Fig. 7 is, perhaps, similar to that found by Schmidt & Stampfli (1966) and Koppenh6fer (1967). These authors also found effects of TEA on the kinetics of the sodium conductance. The possibility that TEA might have a similar action in skeletal muscle has not been examined, because of the difficulties in clamping the sodium current over much of the potential range used in the present experiments. But it seems quite possible that TEA might have some action here, not only by analogy with the work of Koppenh6fer (1967) on Xenopus node, but also because Hagiwara & Watanabe (1955) demonstrated that

19 TEA AND MUSCLE DELA YED RECTIFICATION 227 TEA was able to substitute partly for sodium in the generation of the action potential. A further point of some interest in the complex time course of inactivation of the outward current and the presence, as described by Adrian et al. (1970b), of a second potassium channel whose conductance changes more slowly with time and which has an equilibrium potential closer to the resting potential than does the delayed rectifier. The demonstration of TEA resistance shows that the slowly activating conductance change cannot be a manifestation, on depolarization, of the same process as the slow inactivation of potassium current through the inward rectifier obtained when the membrane is hyperpolarized. The results of the following paper (Stanfield, 1970) show that TEA does block currents through the inwardly rectifying channel. A similar conclusion as to the difference of the slowly activating potassium channel and the inward rectifier has been reached on other grounds by Adrian et al. (1970b). It is noteworthy that the delayed current of the puffer (Spheroides maculatus) is also complex (Nakajima & Kusano, 1966) but that in this case the slower phase of activation is the more strongly affected by TEA (Nakajima, 1966). A point of more general interest concerns possible mechanisms for mechanical activation. Several authors (Costantin, 1968; Kao & Stanfield, 1968; Heistracher & Hunt, 1969) have described how the thresholds for mechanical activation and delayed rectification lie close together under a variety of conditions, a finding which raised the interesting possibility that there might be some, perhaps, causal relation between the two. Heistracher & Hunt (1969) have argued against this since, in high potassium solutions, contraction can occur when the potassium current is inward through the delayed rectifier. But the conductance changes-the changes within the ionic channel-are presumably much the same whether the potassium equilibrium potential is normal or raised. A stronger argument is that caffeine affects only the mechanical and not the potassium threshold (Lorkovic & Edwards, 1968; Heistracher & Hunt, 1969). The present experiments also provide evidence against any causal link between the two processes. TEA reduces the conductance change to potassium (and apparently slows it) while acting as a potentiator of the twitch. Some other explanation for the parallel movements of the thresholds for mechanical and potassium activation in the presence of calcium (Costantin, 1968) and various anions (Kao & Stanfield, 1968) is therefore required. A suitable explanation may be that put forward by Kao & Stanfield (1970) suggesting that the ionic agents which produce these parallel shifts in thresholds do so by altering the membrane surface charge (see also Frankenhaeuser & Hodgkin, 1957; Hodgkin & Horowicz, 1960b).

20 228 P. R. STANFIELD The author wishes to thank Professor A. L. Hodgkin, Dr R. H. Adrian and Dr S. Nakajima for much helpful discussion during the course of the work and for their comments on the manuscript; also Mr R. H. Cook and Mr W. Smith for invaluable technical help; and the Medical Research Council for a Scholarship during tenure of which the work was carried out. REFERENCES ADRIAN, R. H. (1956). The effect of the internal and external potassium concentration on the membrane potential of frog muscle. J. Phy8iol. 133, ADRIAN, R. H., CHANDLER, W. K. & HODGKIN, A. L. (1966). Voltage clamp experiments in skeletal muscle fibres. J. Phy8iol. 186, 51-52P. ADRIAN, R. H., CHANDLER, W. K. & HODGKIN, A. L. (1968). Voltage clamp experiments in striated muscle fibers. J. gen. Phy8iol. 51, ADRIAN, R. H., CHANDLER, W. K. & HODGKIN, A. L. (1970a). Voltage clamp experiments in striated muscle fibres. J. Physiol. 208, ADRIAN, R. H., CHANDLER, W. K. & HODGKIN, A. L. (1970b). Slow changes in potassium permeability in skeletal muscle. J. Phy8iol. 208, ARMSTRONG, C. M. & BINSTOCK, L. (1965). Anomalous rectification in the squid giant axon injected with tetraethylammonium chloride. J. gen. Physiol. 48, AsKARi, A. (1966). Uptake of some quaternary ammonium ions by human erythrocytes. J. gen. Phyaiol. 49, CosTANTIN, L. L. (1968). The effect of calcium on contraction and conductance thresholds in frog skeletal muscle. J. Phy8iol. 195, EDWARDS, C., RITCrE, J. M. & WILKIE, D. R. (1956). The effect of some cations on the active state of muscle. J. Phy8iol. 133, FRANXENHAEfUSER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical properties of squid axons. J. Phy8iol. 137, HAGIWARA, S. & SAITO, N. (1959). Voltage-current relations in nerve cell membrane of Onchidium verrwculatum. J. Phyriol. 148, HAGIWARA, S. & WATANABE, A. (1955). The effect of tetraethylammonium chloride on the muscle membrane examined with an intracellular micro-electrode. J. Phy8iol. 129, HEISTRACHER, P. & HuNT, C. C. (1969). The relation of membrane changes to contraction in twitch muscle fibres. J. Physiol. 201, HLnaT. B (1967). The selective inhibition of delayed potassium currents in nerve by tetraethylammonium ion. J. gen. Phy8iol. 50, HILuE, B. (1968). Pharmacological modifications of the sodium channels of frog nerve. J. gen. Phy8iol. 51, HODGKIN, A. L. & HoRowIcz, P. (1960a). Potassium contractures in single muscle fibres. J. Physiol. 153, HODGKIN, A. L. & HOROWICZ, P. (1960b). The effect of nitrate and other anions on the mechanical response of single muscle fibres. J. Phy8iol. 153, HODGKIN, A. L. & HuIxNy, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Phy8iol. 117, HowART=, J. V. (1958). The behaviour of frog muscle in hypertonic solutions. J. Phy8iol. 144, KAO, C. Y. (1966). Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena. Pharmac. Rev. 18, KAO, C. Y. & STANFIELD, P. R. (1968). Actions of some anions on electrical properties and mechanical threshold of frog twitch muscle. J. Phy8iol. 198,

21 TEA AND MUSCLE DELA YED RECTIFICATION 229 KAo, C. Y. & STANFIELD, P. R. (1970). Actions of some cations on the electrical properties and mechanical threshold of frog sartorius muscle fibers. J. gen. Physiol. (in the Press). KOPPENH6FER, E. (1967). Die Wirkung von Tetraathylammoniumchlorid auf die Membranstrome Ranvierscher Schniirringe von Xenopus laevis. Pflugers Arch. ge8. Physiol. 293, LORKOVIC, H. & EDWARDS, C. (1968). Threshold for contracture and delayed rectification in muscle. Life Sci. Oxford 7, LUTTGAU, H. C. & NIEDERGERKE, R. (1958). The antagonism between Ca and Na ions on the frog's heart. J. Physiol. 143, NAKAJrMA, S. (1966). Analysis of K inactivation and TEA action in the supramedullary cells of puffer. J. gen. Physiol. 49, NAKAJIMA, S. & KusANo, K. (1966). Behaviour of delayed current under voltage clamp in the supramedullary neurons of puffer. J. yen. Physiol. 49, RAVENT6s, J. (1937). Pharmacological actions of quaternary ammonium salts. Q. JZ exp. Physiol. 26, SANDow, A. (1965). Excitation-contraction coupling in skeletal muscle. Pharmac. Rev. 17, SCHMIDT, H. & STAMPFLI, R. (1966). Die Wirkung von Tetraathylammoniumchlorid auf den einzelnen Ranvierschen Schnurring. Pfluyer's Arch. ge8. Physiol. 287, STANFIELD, P. R. (1969). The effect of the tetraethylammonium ion on the inwardly rectifying potassium channel of frog sartorius muscle. J. Physiol. 200, 2-3P. STANFIELD, P. R. (1970). The differential effects of tetraethylammonium and zinc ions on the resting conductance of frog skeletal muscle. J. Physiol. 209, TASAXI, I. & HAGIWARA, S. (1957). Demonstration of two stable potential states in the squid giant axon under tetraethylammonium chloride. J. yen. Physiol. 40, WAsHIo, H. & MASHIMA, H. (1963). Effects of some anions and cations on the membrane resistance and twitch tension of frog muscle fibre. Jap. J. Physiol. 13,