ENSAYOS DE SIMULACIÓN DE DAÑO DE FORMACIÓN

3.1.1 Isolation o f the sodium current

The sodium current was isolated in two experiments by subtracting currents in Ringer solution containing 300 nmol/1 TTX from those in Ringer solution (Figure 5, lower right). In one fibre which was used for quantitative analysis, the effect of series resistance (see Chapter 7) was minimised by reducing the Na"^ current with 3 nmol/1 TTX. Sodium currents from this axon are shown in the left panel of Figure 7. The total current in this axon, and the current in 300 nmol/1 TTX, have already been presented in Figure 5.

The peak sodium currents, in normal Ringer and in the presence of 3 nmol/1 TTX, are shown plotted against pulse potential in the upper right panel of Figure 7. An ohmic conductance, as measured for single Na"^ channels between -70 and +20 mV (see Chapter 4, Figure 2), does not fit the measurements at potentials positive to the Na"*" equilibrium potential Enu- Currents at potentials near E^a can be fitted with the Goldman-Hodgkin-Katz (GHK) current equation (Chapter 7, Equation 3), but rectification is stronger than that of the GHK equation at potentials more than about 20 mV positive to E^a- The GHK equation was used in the quantitative analysis in Chapter 7, although it does not correspond to any likely physical model of ion permeation (Hille 1992), because it is a better empirical approximation to the data than an ohmic conductance.

The Na"^ equilibrium potential shifted between the two sets of recordings in the left panel of Figure 7. It is likely that some Na"^ which had accumulated during dissection was gradually lost again, following the cutting of the internodes in pools C and E in “axoplasmic” solution with low [Na^]. Et^a before applying TTX was +19 mV, corresponding to [Na"^]i = 72 mM; after applying TTX, about 12 minutes later, E^a

was +32 mV, corresponding to [Nsf]i = 43 mM.

3.1.2 Voltage dependence o f activation

As explained in Chapter 4 (section 2.1.1), attempts to separate the activation and inactivation processes impose model-based assumptions on the data. This will be done explicitly in Chapter 7, but here, as for the Na"^ currents in large multi-channel patches, the voltage dependence of the peak Na"^ current will be used to derive the voltage dependence of activation. The peak currents in the upper right panel of Figure 7 were divided by the Na^ driving force {E - E^a), to obtain the Na'^ conductance at the peak of the current. The maximum Na"^ conductance in this node was 833 nS, corresponding to 64,000 open channels. The voltage dependence of

activation is shown in the lower right panel of Figure 7, and can be compared with those in Figures 3 and 4 of Chapter 4, which show the same measurements in multi­ channel patches. This comparison will be explored in detail in Chapter 8.

- 1 2 8 m V +52nriV —8 8 m V I (nA) 2 0 n A + 5 9 m V ■ 1 0 ^ y r50 ^ 100 E (mV) L-50 - 1 2 1 m V 2 0 n A - 8 1 m V 5 m s 1 0 0 0- - 1 0 0 - 5 0 0 E (mV) F i g u r e 7

(upper left) Na^ current in a human axon (from Figure 5). Fibre 92D 18.

(lower left) Na"^ current in the same axon, reduced by the addition o f 3 nmol/1 TTX. Note that Ehoia was adjusted from - 8 8 mV (upper) to -81 mV (lower) between these recordings; the pulses relative to Eh„id were the same.

(upper right) Amplitudes o f peak Na"^ currents in the same axon, plotted against the potential of the test pulse. Filled circles: peak currents in Ringer solution (currents in the upper left panel); open circles: peak currents after the addition o f 3 nmol/1 TTX (currents in the lower left panel).

(lower right) The voltage dependence o f the peak Na'^ conductance, calculated by dividing the peak currents in the upper right panel by the driving force E - E^a- The smooth lines are fits to the Boltzmann function:

^ Na ^ Na max/ ( l + e x p ( ( £ „5- £ ) / i ) )

where G^amax is the total nodal Na"^ conductance. The values obtained from the currents in the presence o f 3 nmol/1 TTX were: F0.5 = -4 5 .3 mV; = 5.5 mV.

Gsamzx without TTX was 833 nS. Filled and open circles have the same meaning as

in the panel above.

The Na^ current activates much more steeply in the absence of TTX than in 3 nmol/1 TTX. In other axons, activation was even steeper. This phenomenon can be

completely explained in terms of the resistance in series with the node, and will be explored in Chapter 7.

3.1.3 Voltage dependence o f inactivation

Depolarising prepulses of 50 ms duration produced voltage-dependent steady-state inactivation of the sodium current (left panel of Figure 8). A test pulse to around -10 mV, immediately following the prepulse, was used to measure the remaining Na"^ current. The peak amplitude of the remaining Na"^ currents (normalised to that found with complete removal of inactivation) is shown for 4 axons in the right panel of Figure 8. Measurements in some axons show a deviation from the fitted Boltzmann

-8mV pre lOnA 2ms I z OH---r - —150 —100 - 5 0 0 E (mV)

Figure 8

(left) Na"^ currents elicited by a pulse to -8 mV, following 50 ms prepulses to between -118 and -48 mV {E pre) to inactivate the Na"^ current. Fibre 92D18, in Ringer solution containing 3 nmol/1 TTX.

(right) Voltage dependence of steady-state inactivation in 4 fibres, measured from currents like those in the left panel. The points were normalised by fitting the Boltzmann function:

/ — I N a Na max

and dividing all points by I^a max- The following values were obtained from the fit: Eos = -80.6 mV; A: = 9.2 mV. In two axons, where the absolute potential was measured in isotonic KCl, the potentials where 30% of the Na"*" current was inactivated were -87 mV and -89 mV. Points from these axons are shown at their actual potential. The points from the other two axons were fitted to the Boltzmann function above, and the potential adjusted so that I^a / max = 0.7 at -88 mV. Fibres 92D18, A2D18 and B2916 (symbols as in Figure 6) and D2N02 (stars).

equation at the most negative potentials, which is identical to that found by Brismar (1980), and was explained by him as being due to the removal of slow inactivation by extreme hyperpolarisation. Assuming this to be the correct explanation, these points were ignored in the Boltzmann fit so that it would represent only fast inactivation.

This figure can be compared with Figures 5 and 6 o f Chapter 4, which show Na"^ channel inactivation in multi-channel patches.

3.1.4 Time course o f inactivation

At potentials more than about 25 mV positive to the holding potential, the time course of inactivation was measured from the falling phase of the Na"^ current during a depolarising pulse. At some potentials, two exponential functions rather than one were needed to describe Na"^ current inactivation during a depolarising pulse. The slower component was larger at more negative potentials, and disappeared at potentials positive to 0 mV. Similar behaviour has already been described in multi­ channel sodium currents (see Chapter 4). The faster component of inactivation was eliminated by adding a low concentration of TTX (3 nmol/1) to the Ringer solution superfusing the node. The time constants resulting from fitting two exponential functions to the decay of Na^ currents in one node are shown in Figure 9.

- 5 0 0 50

Figure 9

Slow (open circles) and fast (filled circles) time constants of Na"^ current inactivation during depolarising pulses. The decay of the currents was fitted with the sum of two exponential functions. Fibre 92D18.

E (mV)

The time course of development of Na"^ current inactivation at potentials near to the holding potential was measured using the three-pulse protocols illustrated in Figure 10. A 50 ms hyperpolarising pulse was used to remove inactivation, followed by a pulse of variable duration to induce inactivation, and a depolarising pulse to determine how much Na"^ current could still be activated (Figure 10, left panel). The dependence of the peak Na"^ current on the duration o f the second pulse (the pulse which induced Na"^ inactivation) could be fitted with a single exponential function of time. An analogous pulse protocol was used to measure the time course o f removal of inactivation (Figure 10, right panel).

3.1.5 Time course o f activation and deactivation

Na"^ current activation followed a sigmoid time course at most potentials, and could be described by a single exponential preceded by a delay. The delay became

the opposite o f the potential dependence for this delay found in most preparations (Patlak 1991); the discrepancy could be due to an imperfect voltage clamp at the onset of the Na"^ current in the present study, or to a difference between human axonal and other Na"^ channels. The first seems more likely. The time course of Na"^ current activation is therefore not considered further here; in the quantitative analysis, it was derived from the time to peak of the Na"^ current, and not from the current during the first few milliseconds of the pulse (see Chapter 7).

-1 4 2 m V lOnA —12mV jE D T J u :___[ ___[ ___r I - 9 2 m V 20ms - 5 2 m V - ^ T J J J U l 1CnA —12mV _[___[ ___T -92mV 20ms

Figure 10

(left) Time course of the onset of Na"^ current inactivation at -92 mV. Following 50 ms prepulses to -142 mV to remove inactivation, pulses of various durations to -92 mV were applied followed by brief pulses to -12 mV to measure the amount of Na"^ current which could still be activated. Fibre 92D18.

(right) Time course of the removal of Na"^ current inactivation at -92 mV. The pulse protocol was identical to that in the left panel, except that the -92 mV pulses were preceded by 50 ms prepulses to -52 mV, to fully inactivate the Na"^ current.

The time course of deactivation of Na'^ currents at potentials near the holding potential was measured from “tail currents”, when the membrane was stepped to various negative potentials after a short depolarising pulse to maximally activate the sodium current (Figure 11).

-12mV -132m V lOnA -52m V -102mV 1ms

Figure 11

Na"^ “tail” currents; deactivation of Na"^ currents at potentials between -102 mV and -52 mV, following activation by 0.2 ms pulses to -12 mV. Fibre 92D18.