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