CH3 (CH2)njN+ (CH3)3 (Cn TMA) on the sodium current in giant

J. Physiol. (1985), 361, pp. 47-64 47With 12 text-figures

Printed in Great Britain

DUAL EFFECTS OF INTERNAL n-ALKYLTRIMETHYLAMMONIUM IONSON THE SODIUM CURRENT OF THE SQUID GIANT AXON

BY J. R. ELLIOTT, D. A. HAYDON AND B. M. HENDRYFrom the Physiological Laboratory, Downing Street, Cambridge CB2 3EG

and the Laboratory of the Marine Biological Association, Plymouth PLi 2PB

(Received 1 August 1984)

SUMMARY

1. The actions of members of the homologous series of alkyl cationsCH3 (CH2)nj N+ (CH3)3 (Cn TMA) on the sodium current in giant axons of Loligoforbesi have been investigated. The substances tested correspond to n = 6, 8, 10, 12,14 and 16.

2. These cations only produced significant sodium current suppression whenapplied inside the axon. Actions on first-pulse sodium currents and use-dependenteffects were separately studied.

3. The shorter members of the series (C6TMA and C8TMA) produced suppressionof first-pulse sodium currents without causing significant use dependence. Thefirst-pulse suppression arose partly from a positive shift along the voltage axis of thesteady-state activation parameter (mxO ) and partly from a reduction in the maximumsodium conductance (aNa).

4. C12TMA and C14TMA produced little first-pulse suppression but caused clear usedependence. Cj0TMA showed intermediate properties while C16TMA was inactive.

5. The use-dependent actions have been quantitatively investigated using adouble-pulse protocol. The results are consistent with a model in which the cationsenter a blocking site on the ion-channel via the intra-axonal aqueous phase. Thecations appear able to bind to inactivated sodium channels at significant rates.

6. The possible molecular locations of the sites responsible for m. shifts and usedependence are discussed. It is argued that the existence of two separate sites mayhelp to explain certain distinctions between the actions ofneutral general anaestheticsand clinical local anaesthetics on the sodium channel.

INTRODUCTION

A number of studies have been concerned with the actions of a range of neutralanaesthetics on the sodium current in squid giant axons (Haydon & Urban, 1983a-c;Haydon, Elliott & Hendry, 1984). It was often found that sodium current suppressionoccurred largely through a positive shift along the voltage axis of the steady-stateactivation parameter (mw). Recently, it was demonstrated that internal applicationofn-octyltrimethylammonium ions (C8TMA) also shifted mo, in the positive direction(Elliott, Haydon & Hendry, 1984). It has been suggested that m. effects may occur

J. R. ELLIOTT, D. A. HA YDON AND B. M. HENDRY

through anaesthetic adsorption at the internal lipid/aqueous interface and may bethe result of alterations in the internal lipid dipole potential (Elliott et al. 1984;Haydon et al. 1984). Preliminary experiments on n-dodecyltrimethylammonium ions(C12TMA) in squid axons indicated that internal application produced clear usedependence in the sodium current but little first-pulse suppression. The actions ofCTMA and C12TMA appeared quite different, although both were only able tosuppress sodium current when applied intracellularly. These differences between twomembers of an homologous series prompted a more thorough investigation of theseries. The results presented in this paper concern the cations C6TMA, C.TMA,CIOTMA, C12TMA, C14TMA and C,6TMA.The internal effects of these cations in CsF-perfused giant axons have been

separated into actions on the first-pulse sodium current and use-dependent actions.The sodium current records were analysed using equations similar to those proposedby Hodgkin & Huxley (1952). The use dependence was investigated by the use ofa double-pulse protocol (Cahalan, 1978) and is interpreted with a model in which ablocking site inside the ion channel is only accessible to cations from the intra-axonalaqueous phase (Armstrong, 1969; Strichartz, 1973; Hille, 1977). There appear to beclear differences between the sites responsible for m. shifts and for use-dependenteffects.

METHODS

Giant axons were dissected from the mantles of freshly killed Loligoforbesi. The axons were finelycleaned and were usually between 600 and 1000 jsm in diameter.The external bathing solution for experiments with intact axons was of the following composition

(concentrations in mM): NaCl, 430; KC1, 10; CaCl2, 10; MgCl2, 50; Trizma base, 10. The pH wasadjusted to 7-4 by the addition of HC1. All experiments involving internal application of testsubstances were performed on CsF-perfused axons. For these axons, the external NaCl concentrationwas reduced to 107-5 mm and 322-5 mM-choline chloride added. Sodium currents were completelysuppressed where necessary by addition of 0 3 /sM-tetrodotoxin (TTX). The internal perfusate wasof composition (mM): CsF, 345; sucrose, 400; NaCl, 5; HEPES, 10. The pH was adjusted to 7 3by the addition of Cs2C00.

Details of the chamber in which the axons were mounted, the electrodes, the perfusion techniqueand the means of introducing the external bathing solution have been described previously(Haydon, Requena & Urban, 1980). The perfusion capillary had an external diameter of approx-imately 450 ,um. In order to change the internal perfusate from a control to a test solution, or viceversa, the axon was reperfused by at least two insertions of the capillary. The voltage-clamp anddata-acquisition procedures were as in Kimura & Meves (1979) and the analysis of the sodiumcurrents was as described by Haydon & Kimura (1981).Compensation for the series resistance was applied in all experiments. To examine the possibility

of artifacts occurring due to incomplete series resistance compensation, sodium current suppressionby low concentrations of TTX was analysed. The results indicated that the maximum error inassessing voltage shifts of m. in a perfused axon with 50% suppression of sodium current was< 1 mV. The experiments were carried out at 6+1 'C. The n-alkyltrimethylammonium ions weresynthesized as bromides. Some of these were kindly donated by Dr R. Klein.

RESULTS

External applicationIntact axons were employed the resting potentials of which were in the range -50

to -60 mV. They were voltage clamped to -55 or -60 mV. Prior to the 15 msdepolarizing test step, a 50 ms pre-pulse to -80 mV was applied to remove fast

48

DUAL ANAESTHETIC MECHANISMS IN NERVE

inactivation. The test pulse for measurement of peak inward current (Ip) was thatwhich gave the maximum current under control conditions. This pulse was usuallyto -10 or 0 mV. External application of 8.0 mM-C6TMA caused a reversible 10-15%increase in peak inward current. There was also a small (1-2 mV) positive shift inthe voltage at which peak inward current was stimulated. Similar small increases insodium current have been reported for external application of 0-5-5 0 mM-C8TMA(Elliott et al. 1984). External application of 100 sM-COTMA to an intact axon causeda reversible 14% increase in peak sodium current without an obvious shift in the peakof the current-voltage relationship. C12TMA applied externally at 12-5 JtM had noeffect on the sodium current; 100 ,tM-C12TMA caused a 10% increase in current whichwas not reversible. Application of 25 /sM-C14TMA had no reversible effect on thesodium current but caused a small irreversible suppression of current.

First-pulse effects of internal applicationCsF-perfused axons were voltage clamped at -70 mV and a pre-pulse of 50 ms

duration to -90 mV was employed. The test depolarizing stimuli were of 15 msduration. In the presence of significant use dependence it was important to use timeintervals between stimuli long enough to allow recovery from block between pulses.C6TMA and C8TMA showed little use dependence and intervals of 2-5 s were foundto be satisfactory. For CjOTMA and C12TMA intervals of 5 and 7 s respectively weresufficient for recovery. For C14TMA and C16TMA intervals of 7 s were used despitethe fact that as much as 15 % increase in block was observed after a normal familyoffifteen stimuli. Longer intervals were not employed as they increased the time takenfor acquisition ofdata beyond the usual survival time for an axon. The effects of theseions on first-pulse data were therefore slightly over-estimated by these experiments.However, the observed actions of C14TMA and C16TMA were small.The final records in each experiment were obtained in the presence of TTX. These

TTX-insensitive currents were subtracted prior to the analysis of sodium currents.The sodium currents were analysed as in earlier work (Haydon & Urban, 1983 a)according to equations derived from the relationships of Hodgkin & Huxley (1952),i.e.

INa = I4a [1-exp (-t/Tm)]3 [hoo (1 -exp (-t/lr))+exp (-ti^h)], (1)INa = m9Na (V- VNa). (2)

This analysis allows the effects on sodium current to be dissected into the alterationsin the Hodgkin-Huxley parameters. The steady-state inactivation parameter, h.,was determined by applying 50 ms pre-pulses at various voltages followed by a testpulse sufficient to give approximately the maximum sodium current. In Table 1appears a summary of the first-pulse data. This includes the effects on peak inwardcurrent (Ip), m., ho, the activation and inactivation time constants (Tm and rh) and9Na. The figures for Ip and 9Na are means of the fractional suppressions. The figuresfor mc, and ho, are means of the voltage shifts in the mid-point of each curve (A Vmand A Vh respectively). The figures for Tm are the fractional change of the peak value.When the sodium currents were use dependent the values of 7h near its peak weredifficult to obtain. The figures in Table 1 for Th are the fractional change of the valueat a membrane voltage of 0 mV.

49

J. R. ELLIOTT, D. A. HAYDON AND B. M. HENDRY

In some axons, in order to obtain accurate subtraction of the TTX-insensitivecurrents, no control data were obtained. In such cases the first-pulse effects on Ip, GNagmo. and hC, were quantified by using mean values obtained from other axons in controlconditions. The figures in Table 1 which include contributions from experiments ofthis type are marked with an asterisk.

TABLE 1. The effects of some intra-axonal alkyl cations on first-pulse sodium currents inCsF-perfused axons

Conen. AVm ATVh No. ofSubstance (mM) I /Ip 9 a/gNa (mV) (mV) t/tm Ti/Th axonsC6TMA 8-0 0-36 0-57 6-6 0 9 0 74 0-88 2C8TMAt 0'8 0.51 0 77 6-7 0.1 0-66 0-83 8C1OTMA 0-08 0 75 0-85 3-1 0-2 0 79 0 93 3CIOTMA 0.15 043 0.62* 6.4* 0 1 - 4C12TMA 0-0125 0-87 0-86 0-8 0-7 0-89 0 99 2C,2TMA 0-025 0-81 0.84* 1.6* 1.2* 0 9 0-92 5C14TMA 0-005 0-8 0-8* 0.1* 1C14TMA 0-025 1.0* 1.0* 0.2* 0.5* 1C16TMA 0.015 1.0* 1.0* 0.0* 0-2* - 1

The superscript t refers to test conditions. Peak inward current (Ip) and maximum sodiumconductance (9Na) are expressed as the mean ratio of test to control values. Average voltage shiftsin the mid-points ofmO0 and h. are designated as A Vm and A Vh respectively. Tm values are comparedat the peak of the curve relating Tm to membrane voltage, rh values are compared at zero membranevoltage.

t From the results of Elliott et al. (1984.) The asterisks indicate that not all the experimentsincluded control results as explained in the text.

The effects of 8'0 mM-internal C6TMA on the first-pulse I'a- V relationship in oneaxon are illustrated in Fig. 1. The suppression of current is associated with a positiveshift in the voltage at which peak current occurs. The corresponding effects on theHodgkin-Huxley parameters are shown in Fig. 2. There is a positive shift of the m".curve along the voltage axis of about 7 mV, but no significant effect on h.. Both Thand Tm are slightly reduced. The value of 9Na in this axon was 17-6 mS cm-2 in thepresence of internal C6TMA and increased to 27-5 mS cm-2 after its removal. Theseactions are similar to those reported for 0-8 mM-internal CTMA (Elliott et al. 1984).As for C8TMA the sodium current suppression by C6TMA arises largely from effectson mo, and 9Na-None of the alkyl cations tested produced significant changes in the h. curve. As

seen in Table 1 the values ofTm and Th, where measured, were reduced in the first-pulsecurrents by the internal cations. These concentrations in the bulk phase were chosento correct approximately for the different membrane/aqueous solution partitioncoefficients of the various compounds. They are expected to have resulted in aboutthe same drug concentrations in the axon membrane (see later). At the concentrationsemployed, the effects on m. and 9Na declined with increasing chain length. 12-5 ,UM-internal C12TMA had no significant effect on mo,. 25 ftM-internal C14TMA and15 /tM-internal C16TMA appeared to have no action at all on the first-pulse sodiumcurrents.

50

DUAL ANAESTHETIC MECHANISMS IN NERVE 51

0*5/I .(mA cm-2)

-50 0 50 100

C; * V (mV) *

0~~~~* -0-5 0-

0

-1-0 0

o 0c1-5

o 0

00 -2-0

Fig. 1. The effects of 8-0 mM-internal CTMA on the I' -V relationship in a CsF-perfusedaxon. V is the membrane voltage. The control data (0D) were obtained after the test data(-) following removal of C6TMA. Axon 618.

1-0 o ° 0 °0 1.0 e0 9 CP 0OO*0.

0

00

Q0-5 0.0 8Q.5

00

0~~~~~~~~~~~~

0.9 O 9 0 °0000

0 0

E 0-6 0 . 0 6E .o E

* 00 0

00-3 3

-50 0 50 -100 -50 0V (mV) V (mV)

Fig. 2. The effects of 8-0 mM-internal C6TMA on various Hodgkin-Huxley parameters. Testdata (@) were obtained before control data ((D). Axon 618.

Use dependence on internal applicationA qualitative description of use dependence was obtained using trains of voltage-

clamp stimuli. Each 15 ms depolarizing test pulse to -10 or 0 mV was preceded bythe usual 50 ms pre-pulse at -90 mV. Internal C12TMA at 12-5 /LM, produced clearuse dependence as shown in Fig. 3. Here, the peak sodium current produced by eachtest pulse is plotted against the number of the pulse in a train of stimuli. In control

J. R. ELLIOTT, D. A. HA YDON AND B. M. HENDR Y

conditions (open circles) there is little difference between the sodium currents elicitedby the first and tenth stimuli of a 10 Hz train. In the presence of internal C12TMAat 10 Hz the tenth-pulse current is reduced to 56% of the first-pulse current. Resultsobtained in the presence of 8-0 mM-internal C6TMA are indistinguishable from thecontrol data in Fig. 3.

1-0 - 01~0 * ~0 0 a a

0a a a

A

AAA

A AAA A A

0-5~~~~~

/~~0* *f,*,

0 1 2 3 4 5 6 7 8 9 10No. of pulse

Fig. 3. Use dependence of the sodium current caused by 12-5 jfM-internal C12TMA. Theordinate is the maximum inward current produced by each pulse in a train of stimuliexpressed as a fraction of the current elicited by the first pulse. Control data obtainedat 10 Hz (()) are compared with results obtained in the presence of C12TMA at 5 Hz (-)and at 10 Hz (@). Axon 523.

A more quantitative analysis of use dependence was attempted using the double-pulse protocol illustrated in Fig. 4A. The holding potential was usually -70 mV andthe first pulse (PI), was a depolarizing step of A V, mV which lasted t1 ms. There wasthen a delay of d ms at the holding potential before a second depolarizing pulse (P2)of magnitude A V2 lasting 15 ms. The first pulse was a conditioning step prior to themeasurement of the current elicited by P2. Experiments were performed to measurethe peak second-pulse current (Ip 2) as a function of tj and d. For these measurements,AVI was set at 160 mV and A V2 at 60 mV. Fig. 4B shows the effect of varying t1 from0 to 70 ms with a constant value of d of 50 ms. The open circles are control data andthe filled circles are data obtained in the presence of 25 ,tM-internal C12TMA. The linedrawn through the data is a single exponential with a time constant of 22-7 ms. Thisline provides a good fit to the results for tj > 2 ms. Fig. 4C shows the variation inIp 2 as d was altered from 2 ms to 2 s. The value of t1 was fixed at 15 ms. In controlconditions (0), this procedure revealed the recovery rate from fast inactivation at-70 mV. When 25 /LM-internal C12TMA was present there was an additional slowcomponent to the recovery of Ip 2. The line drawn in Fig. 4 is a single exponentialwith a time constant of 2- 1 s. It provides a satisfactory fit to the results for d > 50 ms.

This second component of recovery in the relationship between Ip 2and d was seenfor all homologues tested between C8TMA and C14TMA. A comparison of results forC8TMA, C1OTMA and C12TMA is shown in Fig. 5. The rate of recovery appears to be

52


P1 P2l m><15ms

A V A _ i\j V2e.- t,---- d --jo

B 1\ v /P. 2

1-0 Cs 0 0 o 'O 0 C) 0

s ~~~~AV, = 160 mV\ ~~~~~d=50ms\, ~~~AV2=60mV

0 5 25 pM-internal C12TMA

0 25 50 75

C t (iMs)1.0 *OOOO00 000 0

25,MM-internal C12TMA

tj = 15 ms

0 500 1000 2000d (ms)

Fig. 4. A is a schematic diagram of the double-voltage-pulse protocol used to investigateuse dependence. PI is of magnitude AV, and duration t,. P2 occurs after a delay (d) andis ofmagnitude A V2 and duration 15 ms. B shows the effects of varying tj on Ip, 2 in controlconditions (()) and in the presence of 25/M-internal C12TMA (@). A V1 was 160 mV, dwas 50 ms and A V2 was 60 mV. The holding potential was -70 mV. 'p,2 is normalizedand expressed as a fraction of its value for t, = 0. The line is drawn according to therelationship Ip 2 = 0-8 exp (-tl/r) +0X136, with X set at 22X7 ms. Axon 634. C illustratesIp 2 as a function of d in control conditions ((0) and in the presence of 25 #m-internalC12TMA (@). Ip, 2 is expressed as a fraction of its value for d = 7 s. t, was 15 ms and theother parameters were as in B. The line is drawn according to Ip 2 = 1- 037 exp (-d/r),with T set at 2-1 s. Axon 667.

faster for the shorter alkyl chains. For d > 50 ms, satisfactory fits to these resultswere obtained using single exponentials. Mean time constants obtained in this mannerfor each substance are listed in Table 2 and are designated as r (dissociation). Thescatter of the results for r (dissociation) was such that all values were within + 23%of the means quoted in Table 2.Examples of relationships between Ip 2 and t1 are shown in Fig. 6 for C8TMA and

CIOTMA. For C12TMA and C14TMA, the results ofthis type ofexperiment can be fittedwith a single exponential for t1 > 2 ms. The results for C8TMA and CjOTMA are morecomplex and two or more exponentials are required for a good fit. A crude idea of


1.0 -1><0 9 & & *0 ~~ ~ ~ ~ A

A A A A

0 0 A~~~~~0 A0

* A-

A M

A

0 200 400 600 800 1000d (ms)

Fig. 5. A comparison of the relationships between Ip, 2and d in control conditions ((D) andin the intra-axonal presence of0-8 mM-C8TMA (-), 80 /M-C10TMA (-) and 25 1sM-C12TMA(I). In each case A V1 was 160 mV, A V2 was 60 mV and t£ was 15 ms. The holding potentialwas -70 mV. The control data were from axon 684 and the test data were from axons684, 666 and 634 respectively.

TABLE 2. Use-dependent effects of some intra-axonal alkyl cations on the sodium current7 (dis- r (associa-

Conen. sociation) tion) No. ofSubstance (mM) (Ms) (Ms) g2 a/9a AVm,2 r2 /T" r7/Tj axonsCTMA 0-8 57 0-8 0 74 -241 1.0 1*17 2COTMA 0-08 260 5-4 0 34 -0 7 0 77 1-73 3CIOTMA 0.15 298 3 0 0-28 -2-6 0 79 191 4C1,TMA 0-025 2170 21 0 39 0-2 0 77 1-26 4C14TMA 0-025 5800 84 0 77 0 3 0 91 1-04 1C,,TMA 0-015 - 0-92 0 7 0 94 1.0 1

The superscripts 1 and 2 refer to first- and second-pulse results respectively, both obtained inthe presence of the alkyl cation. r (dissociation) values were obtained by fitting data such as thosein Fig. 4C using the same double-pulse parameters as in that example. r (association) values wereobtained as described in the text. For these experiments d was set at 50 ms except when C8TMAwas tested, where d was 30 ms. The alterations in Hodgkin-Huxley parameters between first- andsecond-pulse currents were obtained using the double-pulse parameters of Fig. 8. A Vm, 2 is the meanvoltage shift of the mid-point of the m., curves obtained from first-pulse and second-pulse currents.Tm and Th are compared as peak and zero membrane voltage results respectively and expressed asmean ratios of second-pulse to first-pulse values.

the rates at which C8TMA and CIOTMA act during P1 can be obtained by interpolatingfor each substance the value of t1 at which the normalized value of Ip, 2 falls toI00+ 1/e (1-I1), where I.j is an estimated value for Ip,2 at t, = oo. Using thismeasure where necessary, Table 2 shows the mean time constants obtained from therelationships between Ip 2 and t1, where they are designated as r (association). Thescatter of the data for r (association) was such that all values were within ± 20% ofthe means quoted in Table 2.

54


Experiments to investigate the effects on Ip, 2 of varying A V1 were performed witha holding potential of -100 mV. The value ofA V2 was accordingly increased to 90 mVso that the membrane voltage during P2 remained at -10 mV. The results of twoexperiments of this form are displayed in Fig. 7 where IP 2 is plotted against Vpj the

1-0 '0oc O O O

A0* *- *-0 * *A

A

0-5 AA

AA

A AA A

A A

0 25 50 75t1 (ins)

Fig. 6. A comparison of the relationships between IP 2 and t1 in control conditions (0)and in the intra-axonal presence of 0-8 mm-CTMA (@) and 80 lum-CI0TMA (A). In eachcase A 17, was 160 mV and A V2 was 60 mV. For the control data and for CTMA (axon 684)d was 30 ms while for CI0TMA (axon 666) d was 50 ins. The holding potential was -70 mV.

membrane voltage during PI (A V1 -1I00 mV). Fig. 7A shows control data and resultsobtained in the presence of 0-8 mm-internal C8TMA. The considerable first-pulsesuppression with the C8TMA is indicated by the values of I, 2for Vpj more negativethan -60 mV. There is a small additional second-pulse block and Ip, 2 declines withincreasing Vpj to reach a steady level for Vp, > 50 mV. A similar experimentinvolving 0-80 mm-internal Cj0TMA is shown in Fig. 7 B.The second-pulse inhibition is of the same form but here it is slightly larger than

the first-pulse effect. The test data in Fig. 7 show a broad maximum at voltagesaround -60 mV. This was a reproducible finding and could also be discerned in thedata for C12TMA and C14TMA. These longer chain homologues produced resultssimilar to those in Fig. 7 except that IP, 2 continued to decline as Vpj increased to80 mV and beyond. For C12TMA the small first-pulse suppression was dominated bythe second-pulse inhibition. In one experiment, C16TMA was applied inside the axonat 15 fum. The maximum use-dependent suppression was less than 10 %.-The rate of recovery from use-dependent block at a holding potential of -100 mV

was not systematically investigated but it was established that this recovery was notsignificantly slower than at -70 mV. For example, in the presence of 25 gM-internalC12TMA recovery from block at -100 mV was > 95% complete after 7 s.

56 J. R. ELLIOTT, D. A. HA YDON AND B. M. HENDRY

Hodgkin-Huxley analysis of second-pulse currentsIn these experiments the effects of varying A V2 on the second-pulse current were

investigated. The values of t1, A V1, and d were held constant and the holding potentialwas -70 mV. An example ofdata obtained with this protocol is shown in Fig. 8. Theseresults were obtained with 25 /LM-C12TMA inside the axon and the sodium current

A 1-5 B 1-5

0o0oo0000 00 0 0 0

000000000 )0 0 00* -10 1-05

E E

E E

@00000. 0

-100 0 100 -100 0 100

VP, (mV) VpI (mV)

Fig. 7. The relationships between A V1 and Ip 2in control conditions (0) and in the internalpresence of 0-8 mM-C8TMA (*, graph A) and of 80 /sM-CloTMA (*, graph B). The holdingpotential was -100 mV. Vp, is the membrane voltage during the first pulse(VP1 = A V1-100 mV). A V2 was 90 mV, t1 was 25 ms and d was 30 ms for C8TMA (axon684) and 50 ms for C10TMA (axon 666).

is plotted against the membrane voltage VP2 during P2. The open circles (0) are datafor AV, = 0 and correspond to the first-pulse current-voltage relationship in thepresence of C12TMA. The filled circles (@) were obtained with AV1 = 160 mV,t1= 20 ms and d = 50 ms. The current is reduced at all voltages by the presence ofthe first pulse. The membrane voltage at which peak inward current occurs isunaltered.

Records of the second-pulse sodium current have been analysed according to eqns.(1) and (2) to obtain the voltage-dependent Hodgkin-Huxley parameters. The majordifference between first- and second-pulse currents was in the values of 9Na. Forexample the currents used to construct Fig. 8 gave values for -Na of 27-1 mS cm-2(first pulse) and 9-98 mS cm-2 (second pulse). The mid-point of the m. curve was notsignificantly altered. The time constants Tm and Th do appear to differ betweenfirst-pulse and second-pulse currents. An illustration of this effect in the presence ofC12TMA is seen in Fig. 9 for Th. The second-pulse currents exhibit higher values ofTh. Table 2 includes a comparison of the Hodgkin-Huxley parameters for first- andsecond-pulse currents in the presence of various n-alkyltrimethylammonium ions.The values Of 9Na' Tm and Th are compared using the ratios of the second-pulse tofirst-pulse results. Th values are compared at a membrane voltage of zero while the


-50

0 0e

0

0

0

0

0

*

0

0

00

- 04

/ (mA cm 2)

50

-0-4

-0 80

0

0

* 100

V (mV) 0

0 0

0

0

0

- -1 2Fig. 8. First-pulse and second-pulse current-voltage relationships in the presence ofinternal C12TMA at 25 pM. The test data (@) are measurements ofIp,2 with A V1 at 160 mV,t, at 20 ms, d at 50 ms and a holding potential of -70 mV. V is the membrane voltageduring the second pulse (A V2-70 mV). The control data (0) were also obtained in thepresence of C12TMA but with AV1 = 0 so that they represent first-pulse currents. Axon634.

.

0

0

0

0

.

0 *0

000

- 6

- 4 -a

- 2.

-50 0 50V (mV)

Fig. 9. A comparison of Th values obtained by analysis of first-pulse (0) and second-pulsecurrents (@) in the presence of 25/SM-internal C12TMA. The set parameters were as inFig. 8 and the currents analysed are those the maxima of which are plotted in Fig. 8.Axon 634.

peak values of Tm are employed. The mOO results are compared by measurement ofthe voltage difference (A Vm, 2) between the mid-points for second-pulse and first-pulsedata.Where they are significant the m. shifts in Table 2 are negative. For example in

a typical experiment using 0-8 mM-internal C8TMA (axon 684), the control mO,,mid-point was at a membrane voltage of -39-4 mV. In the presence ofCTMA thiswas shifted to -33-5 mV for the first-pulse sodium currents and to -35-5 mV for

l a

-I I

57


the second-pulse currents. The conditioning pulse appeared to shift m"" back towardscontrol values; it did not enhance the positive first-pulse shift of mO.

DISCUSSION

External application of the quaternary ammonium ions employed in this studyproduced little effect on the sodium current. These results are consistent with theeffects of 0-8 mM-external C8TMA reported by Elliott et al. (1984). Significant sodiumcurrent suppression was only obtained following intra-axonal application. The majoreffects of internal n-alkyltrimethylammonium ions demonstrated in this paper wereon the Hodgkin-Huxley parameters mcj. and Na. Use-dependent suppression ofsodium current was largely due to reductions in -Na. There are good reasons tosuppose that the shifts of m. and the use-dependent effects are mediated fromdifferent sites of action. First, the relative importance of the two actions varies withalkyl chain length. C6TMA produced an mOD shift but did not cause significant usedependence; C12TMA caused pronounced use dependence but had little effect on mci.Secondly, the use dependence was enhanced by increasingly positive conditioningdepolarizations while the m. shifts were not made larger by this conditioning.

Possible origins of the m. shiftsThe effects of 8-0 mM-internal C6TMA on mo, described here are very similar to those

reported by Elliott et al. (1984) for 0'8 mM-internal C8TMA. The explanationsproposed in earlier work for these m. shifts appear capable of explaining the datafor C6TMA and C10TMA (Haydon & Urban, 1983b; Haydon et al. 1984; Elliott et al.1984). Essentially, it has been suggested that these amphipathic molecules adsorbat the internal lipid/aqueous interface of the axonal membrane. This adsorptionreduces the lipid dipole potential at that interface and so alters the electric field withinthe membrane. If the activation voltage sensor is a dipole located in the membraneinterior, then this alteration in electric field can account for the observed m., shifts.Whatever their precise molecular origins, the mi0 effects produced by the shorter alkylcations tested here are similar to the actions of a wide range of surface-active neutralgeneral anaesthetics on the squid giant axon (Haydon et al. 1984). This similaritysupports the notion that such neutral molecules affect m,0 from a site at the internalmembrane/water interface.As seen in Table 1, the effects of these alkyl cations on m.0 appear to decline with

increase in alkyl chain length. To assess the importance of this decline someexplanation for the choice of concentrations used is necessary. The initial experimentswere performed with internal C8TMA and a concentration of 0-8 mm was found tocause approximately 50 % reduction in peak inward sodium current. If the alkylcations were acting at an aqueous/hydrocarbon interface then increasing the chainlength by two carbon atoms and reducing the aqueous concentration by a factor ofabout 10 should leave the interfacial concentration of cation unchanged (see, e.g.Tanford, 1980). This rule appears to approximate the relative potencies of certainn-alkanols on the squid sodium current (Haydon & Urban, 1983b). In line with thisreasoning, it was found that 8-0 mM-internal C6TMA caused just over 500 suppressionof peak sodium currents. However, 0-08 mM-C10TMA produced considerably less

58


than 50% first-pulse suppression and C12TMA and C14TMA only produced effects onthe first-pulse current at concentrations well above 0 008 and 0-0008 mm, respectively.The results of Table 1 therefore indicate that in comparison with a bulkaqueous/hydrocarbon interface the m. site of action exhibits a decline in adsorptionas the alkyl cation carbon-chain lengths increase.A decline in anaesthetic potency with increase in alkyl chain length has been

reported in a number of systems. The n-alkanes appear to lose anaesthetic potencyfor homologues longer than n-octane (Haydon, Hendry, Levinson & Requena, 1977;Haydon & Hendry, 1982). There is some evidence that the anaesthetic potencies ofthe n-alkanols decline above n-decanol, both on squid axons (Elliott, 1981; B. M.Hendry, unpublished observations) and in other preparations (Brink & Posternak,1948; Pringle, Brown & Miller, 1981). For the n-alkanes an explanation based on theproperties of the lipid bilayer can account for the chain-length effects (Haydon et al.1977). The decline in the moo shifts produced by the longer alkyl cations may be basedon decreased adsorption at the internal lipid/aqueous interface.

Possible origins of the use-dependent suppressionThe use-dependent suppression of sodium current observed in the present work

is similar to that reported for a variety of cationic and ionizable substances in frognode of Ranvier and squid axons (Strichartz, 1973; Courtney, 1975; Hille, 1977;Cahalan, 1978). There are also parallels with the earlier work of Armstrong (1969)on the effects of quaternary ammonium compounds on the potassium channel. Asimple model of the blocking reaction for internal cations has been used to describethese earlier results and this provides a useful framework for analysis of the datadescribed here. The model includes a blocking site within the ion pore of the sodiumchannel which, for cationic substances, is only accessible from the intra-axonalaqueous phase. Movement to and from this site is held to be a function of themembrane voltage and of the state of the channel gates. Using this model the resultsconcerning the relationship between Ip 2and t1 indicate the rate at which the blockingsite is occupied during the first pulse. The data relating Ip, 2to d provide informationabout the rate of unblocking during the delay between pulses. The terms 'association'and 'dissociation' are used in Table 2 to describe these processes.

In Fig. 10 the mean results for r (dissociation) are plotted against the n-alkyl chainlengths of the blocking cations. The straight line is drawn by eye and provides asatisfactory fit to the results for C8TMA, CIOTMA and C12TMA. Extrapolation toCTMA predicts a value of 8-3 ms for r (dissociation). This is close to the time constantfor recovery from fast inactivation at -70 mV. If this extrapolation is correct itexplains why internal CTMA does not produce observable use dependence with trainsof voltage-clamp stimuli at 10 Hz. The rate of dissociation of the bound state at-70 mV may be too fast to be distinguished from the normal repriming of theinactivated channel.The values of r (dissociation) displayed in Fig. 10 represent time constants for the

loss of bound cations at -70 mV; at this potential almost all sodium channels arenormally closed. The relief of block at a holding potential of -100 mV was notsignificantly slower than at -70 mV. The data therefore do not obviously arise froma fast dissociation from a small fraction of open channels present at -70 mV. The

59


closed or resting state of the channel thus appears able to undergo dissociation ofthe bound cation. In this respect the present results are not compatible with the modelused by Hille (1977) and Cahalan (1978) in describing their data on certain otheramphipathic cations.The slope of the line drawn in Fig. 10 is equivalent to a factor of 2-50 per CH2 group.

This slope can be interpreted if certain assumptions are made about the process of

10000

1000 /

0

.X 100o

10

1 II I I 1

6 8 10 12 14No. of carbon atoms

Fig. 10. The relationship between r (dissociation) and the number of carbon atoms in then-alkyl chain of the quaternary ammonium cation present inside the axon. The error barsrepresent the maximum scatter of the results.

dissociation. These are: (1) that one component of the activation energy for thereaction is the energy required to move the alkyl chain from the binding site intoan aqueous environment, and (2) that the variation of r (dissociation) with chainlength is entirely due to alterations in this component of the activation energy.Application of Eyring rate theory (Frost & Pearson, 1961) allows an estimation ofthe standard free energy of transfer per CH2 group for movement from the aqueousenvironment to the blocking site. The line in Fig. 10 gives a value of- 2-13 kJ mol1 CH2 for this energy. This result is consistent with the idea that thebinding site provides a partially hydrophobic environment for the alkyl chain. Asimilar transfer energy of - 2-34 kJ mol- CH2 was estimated by Rojas & Rudy (1976)for binding of intra-axonal n-alkyltriethylammonium ions to pronase-treated squidsodium channels. The transfer energy for adsorption at a bulk aqueous/hydrocarboninterface is about - 3-12 kJ molV CH2 (see e.g. Haydon & Urban, 1983 b). The effectsof n-alkanols on the sodium current can be interpreted to estimate a transfer energyto their site of action of -3-04 kJ mold CH2 (Haydon & Urban, 1983 b). The bindingsite inside the ion channel appears to be more polar than pure hydrocarbon and theseconsiderations lend further support to the idea that it is distinct from the siteresponsible for mO0 shifts.

60


The relationship between r (association) and the intra-axonal concentration ofalkyl cation is illustrated in Fig. I1. These results are difficult to analyse precisely.The time constants for CTMA and C1OTMA are not exponential but have beenobtained by a rather crude interpolation. The results are further complicated by thefact that for t1 < 2 ms a substantial fraction of sodium channels are open, while fort1 > 2 ms the association is essentially with inactivated channels. The line is drawn

100 014

12E s

Z 10 \

(U- 10

10

X~~~~~~~~~~~~

10 100 1000Internal concentration (pM)

Fig. 11. The relationship between -r (association) and the intra-axonal aqueous concen-tration of alkyl cation. The results for CeTMA, C1OTMA, C12TMA and CH4TMA aredistinguished by the figures near each point. The error bars represent the maximum scatterof the results.

in Fig. I I with a slope of -1 and provides a reasonable fit to the data for C8TMA,C1OTMA and C12TMA. This agreement is consistent with a model in which theassociation rate is related to concentration and not a function of alkyl chain length.C14TMA exhibits a slower rate indicating that molecular size may limit the rate forchains longer than twelve carbon atoms. The success of a simple linear fit in Fig. I1suggests that the rates of association to open and to inactivated channels are notvastly different. The results for C12TMA at t, > 2 ms are well fitted by a singleexponential (e.g. Fig. 4). At a membrane voltage of 90 mV inactivation of the sodiumchannel is not complete and 10-30% of channels appear to remain open indefinitely(Chandler & Meves, 1970). It is, therefore, conceivable that one process occurringduring the first pulse is an interaction between the C12TMA and these open channels,but this does not appear sufficient to explain fully the magnitude of the blockobserved. At a membrane voltage of -20 mV steady-state inactivation is practicallycomplete and, for C1OTMA, the idea that the non-inactivating channels contributethe major site of interaction during PI is therefore not supported by the smooth formof the curve in Fig. 7 B. The simplest explanation for the present results is thatC1OTMA and C12TMA can bind to the inactivated form of the sodium channel. For

61


C12TMA this process appears to have first-order kinetics. In the models used by anumber of other workers (Hille, 1977; Cahalan, 1978; Courtney, 1980) intracellularamphipathic cations can only interact directly with the open form of the sodiumchannel. The present results do not support the existence of this restriction for then-alkyltrimethylammonium ions.

R =

0* °*0*R* b

Fig. 12. A model scheme representing the resting (R), open (0) and inactivated (I) statesofthe sodium channel. Asterisks indicate that the n-alkyltrimethylammonium binding siteis occupied. Both 0 and 0* are conducting states. 0* is a blocked state which is onlyaccessible via 0*.

The molecular consequences of binding to the sodium channelIt is widely held that occupation of the binding site responsible for use-dependent

inhibition results in a channel conductance of zero. In the model proposed by Hille(1977) it is further assumed that gating transitions of the blocked channel occur andthat these transitions may differ in their voltage dependence from those occurringin unoccupied channels. In particular, the h. curve for blocked channels is held tobe shifted to negative potentials. It is difficult to test these ideas satisfactorily buttwo features of the present data may be of interest with respect to the molecularconsequences of alkyl cation binding. The first is the observation that Ip declinestowards a non-zero asymptote as A VI increases, as shown in Fig. 7. The second is thatTm and particularly Th appear to differ between first- and second-pulse currents(Fig. 9 and Table 2). If the amphipathic cations undergo a voltage-dependent bindingto a site within the ion channel then at very positive membrane voltages the bindingequilibrium should approach 100% of channels occupied. If dissociation must occurfor a channel to become conducting then Th values for the second-pulse currents mightbe expected to coincide with the first-pulse results.The values of Ip 2 at high A V1 shown in Fig. 7 are elevated by the fact that t1 was

not long enough for equilibrium to occur and by the dissociation which occurs duringthe delay between pulses. However, application of corrections for these factors doesnot reduce Ip 2 to zero. One possible qualitative explanation for these results isillustrated by the scheme in Fig. 12. The possible gating status of each channel issimplified to include just three states: resting (R), open (0) and inactivated (I). Eachof these states can undergo cation binding and the bound forms are designated R*,O* and I* respectively. The O* state is postulated to have the same conductance asthe 0 state and the gating transitions among R*, 0* and I* are identical to those

62


among R, 0 and I. The 0* state can move quickly and reversibly to a blocked state 0band this transition is not voltage dependent. The blocked state can only unblock viathe transition to 0*.The existence of a bound unblocked state 0* explains why Ip 2 does not approach

zero as A V1 increases. The effects on Th may be described by considering the transitionsafter a conditioning pulse has put all the channels into the bound form and the testpulse has removed all the channels from the R* state. Then the possible states arelinked as follows, i.e. Ih fast

ahThe rate constants ash and A3h are those used by Hodgkin & Huxley (1952), so thatthe control rh = 1/(a, +/,8). This is a scheme which is mathematically identical inform to that used by Neher & Steinbach (1978) to describe local anaesthetic blockofacetylcholine-receptor channels. The observed rate ofdecay to the inactivated stateis reduced by the existence of the non-inactivatable state 0*. This scheme is thereforeconsistent with the results showing an increase in rh for the second-pulse currents.It predicts that single sodium channel recordings made during the second pulse inthe presence of internal n-alkyltrimethylammonium ions might show a burstingappearance or, if the 0* ± 0b transitions are very fast, a reduced single-channelconductance in a sub-population of events.An alternative explanation for the data concerning Ip 2at high A V1 and the results

-for rh during P2 is that the test pulse expedites dissociation of blocking cations fromthe sodium channels, the occupied channel having zero conductance. However, thishypothesis predicts that activation of current by P2 should be slowed, whereas thedata of Table 2 indicate that Tm is reduced for second-pulse currents compared withfirst-pulse currents.

Concluding remarksSuppression of sodium current by internal application of n-alkyltrimethyl

ammonium ions occurred through two distinct modes of action whose relativeimportance varied with alkyl chain length. The shorter molecules tested producedshifts in mo, which were similar to those seen in the presence of neutral polar generalanaesthetics such as n-octanol. The longer molecules caused use-dependent inhibitionwhich appears to be due to voltage-dependent binding to a site within the ion channel.This effect is similar to that produced by many clinical local anaesthetics and theircationic derivatives. The precise consequences of binding to this intrachannel site arenot fully understood and a number ofalternative molecular models exist which cannotbe definitively tested at present.

The authors wish to thank Mr Eric Maskell and Mr Peter Webb for valuable technical assistance.J. R. E. acknowledges support from the Medical Research Council. B. M. H. is a Medical ResearchCouncil Training Fellow.

REFERENCES

ARMSTRONG, C. M. (1969). Inactivation of the potassium conductance and related phenomenacaused by quaternary ammonium ion injection in squid axons. Journal of General Physiology 54,553-575.

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J. R. ELLIOTT, D. A. HAYDON AND B. M. HENDR Y

BRINK, F. & POSTERNAK, J. M. (1948). Thermodynamic analysis of the relative effectiveness ofnarcotics. Journal of Cellular and Comparative Physiology 32, 211-233.

CAHALAN, M. D. (1978). Local anaesthetic block of sodium channels in normal and pronase-treatedsquid giant axons. Biophysical Journal 23, 285-311.

CHANDLER, W. K. & MEVES, H. (1970). Evidence for two types of sodium conductance in axonsperfused with sodium fluoride solution. Journal of Physiology 211, 653-678.

COURTNEY, K. R. (1975). Mechanism of frequency-dependent inhibition of sodium currents in frogmyelinated nerve by the lidocaine derivative GEA 968. Journal ofPharmacology and ExperimentalTherapeutics 195, 225-236.

COURTNEY, K. R. (1980). Structure-activity relations for frequency-dependent sodium channelblock in nerve by local anaesthetics. Journal of Pharmacology and Experimental Therapeutics 213,114-119.

ELLIOTT, J. R. (1981). Anaesthetic mechanisms: effects of alcohols on lipid membranes. Ph.D.dissertation, University of Cambridge.

ELLIOTT, J. R., HAYDON, D. A. & HENDRY, B. M. (1984). The asymmetrical effects of some ionizedn-octyl derivatives on the sodium' current of the giant axon of Loligoforbesi. Journal of Physiology350, 429-445.

FROST, A. A. & PEARSON, R. G. (1961). Kinetics and Mechanism, 2nd edn. New York: John Wiley.HAYDON, D. A., ELLIOTT, J. R. & HANDRY, B. M. (1984). Effects of anaesthetics on the squid giant

axon. In The Squid Axon, vol. 22, ed. BAKER, P. F., pp. 445-482. Current Topics in Membranesand Transport. New York: Academic Press.

HAYDON, D. A. & HENDRY, B. M. (1982). Nerve impulse blockage in squid axons by n-alkanes:the effect of axon diameter. Journal of Physiology 333, 393-403.

HAYDON, D. A., HENDRY, B. M., LEVINSON, S. R. & REQUENA, J. (1977). Anaesthesia by then-alkanes: a comparative study of nerve impulse blockage and the properties ofblack lipid bilayermembranes. Biochimica et biophysics acta 470, 17-34.

HAYDON, D. A. & KIMURA, J. E. (1981). Some effects of n-pentane on the sodium and potassiumcurrents of the squid giant axon. Journal of Physiology 312, 57-70.

HAYDON, D. A., REQUENA, J. & URBAN, B. W. (1980). Some effects of aliphatic hydrocarbons onthe electrical capacity and ionic currents of the squid giant axon membrane. Journal of Physiology309, 229-245.

HAYDON, D. A. & URBAN, B. W. (1983a). The action of hydrocarbons and carbon tetrachloride onthe sodium current of the squid giant axon. Journal of Physiology 338, 435-450.

HAYDON, D. A. & URBAN, B. W. (1983b). The action of alcohols and other non-ionic surface activesubstances on the sodium current of the squid giant axon. Journal of Physiology 341, 411-427.

HAYDON, D. A. & URBAN, B. W. (1983c). The effects of some inhalation anaesthetics on the sodiumcurrent of the squid giant axon. Journal of Physiology 341, 429-439.

HILLE, B. (1977). Local anaesthetics: hydrophilic and hydrophobic pathways for the drug-receptorinteraction. Journal of General Physiology 69, 497-515.

HODGKIN, A. L. & HUXLEY, A. F. (1952). A quantitative description of membrane current and itsapplication to conduction and excitation in nerve. Journal of Physiology 117, 500-544.

KIMURA, J. E. & MEVES, H. (1979). The effect of temperature on the asymmetrical chargemovement in squid giant axons. Journal of Physiology 289, 479-500.

NETHER, E. & STEINBACH, J. H. (1978). Local anaesthetics transiently block currents through singleacetylcholine-receptor channels. Journal of Physiology 277, 153-176.

PRINGLE, M. J., BROWN, K. B. & MILLER, K. W. (1981). Can the lipid theories of anaesthesiaaccount for the cutoff in anaesthetic potency in homologous series of alcohols? MolecularPharmacology 19, 49-55.

ROJAS, E. & RUDY, B. (1976). Destruction of the sodium conductance inactivation by a specificprotease in perfused nerve fibres from Loligo. Journal of Physiology 262, 501-531.

STRICHARTZ, G. R. (1973). The inhibition of sodium currents in myelinated nerve by quaternaryderivatives of lidocaine. Journal of General Physiology 62, 37-57.

TANFORD, C. (1980). The Hydrophobic Effect, 2nd edn. New York: John Wiley & Sons.

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Documents

CH3 (CH2)njN+ (CH3)3 (Cn TMA) on the sodium current in giant