Effects of lidocaine on single cardiac sodium channels

J Mol Cell Cardiol 19, 865-874 (1987)

E f f e c t s o f L i d o c a i n e on S i n g l e C a r d i a c S o d i u m C h a n n e l s Bernd Nilius, Klaus Benndorf and Fritz Markwardt

Julius Bernstein Institute of Physiology, Martin Luther University Halle- Wittenberg, Leninallee 6, GDR-4020 Halle (Saale), East Germany (Received 16January 1987, acceptedin revisedform 21 May 1987) B. NILIUS,K. BENNDORFANDF. MARKWARDT.Effects of Lidocaine on Single Cardiac Sodium Channels. Journal of Molecularand CellularCardiology(1987) 19, 865-874. Lidocaine block of single cardiac sodium channels was studied in cell free inside-out patches of ventricular cells isolated from guinea-pig hearts. When applied to the inner surface of the membrane lidocaine depressed Na channel currents by decreasing the probability P of the channels to open measured from the peaks of the averaged currents. In parallel to the decrease in P the relative number of empty sweeps (nulls) was increased. Half maximum block of the activity of single Na channels was observed at 2.9 #M. Lidocaine affected the gating behaviour of Na channels by shortening of the mean open time zo from 0.44 + 0.17 (control) to 0.19 + 0.13 (5 klM lidocaine, holding potential -120 mV, test potential - 6 0 mV). Five micromolar lidocaine completelysuppressed burst-like openings of Na channels and abolished the slow decaying phase of the averaged currents. A shift from -- 120 towards -- 160 mV exerted relieffrom the effect of both Pand z0 .

KEy WORDS: Singleventricular cells; patch clamp ; Na channels; Lidocaine.

Introduction A n i m p o r t a n t group of a n t i a r r h y t h m i c drugs is characterized by their blocking effects on the Na i n w a r d current in myocardial preparations ([13, 16] for a review). A m o n g these drugs lidocaine is widely used in the clinical m a n a g e m e n t of ventricular arrhythmias [2]. However, lidocaine's a n t i a r r h y t h m i c action is not completely understood ([29] for a review). Lidocaine shows the following well k n o w n effects: (i) the peak N a c u r r e n t is reduced in voltage clamped myocardial preparations [1, 21, 28]; (ii) the action potential is shortened [6, 8, 29]; (iii) lidocaine blocks a steady state (window) N a current [5, 6, 8]. I n this study we used cell free inside-out patches from isolated m a m m a l i a n ventricular cells to analyse the lidocaine block of cardiac single Na channels. O u r goal was to collect d a t a on the single c h a n n e l level which can be used to explain these hitherto described macroscopic effects of lidocaine.

Methods All experiments were performed on 200 to 300 g guinea-pigs. After cervical dislocation

the hearts were removed and rapidly m o u n t e d on a L a n g e n d o r f f prefusion apparatus. Ventricular cells were dissociated by a protocol similar to that used by [19]. After dissociation of the heart with 0.1% collagenase (Sigma, type I) a n d 0.1% hyaluronidase (Hylase, Institut f'fir Impfstoffe, Dessau, G D R ) the cells were three times washed with Eagle's M i n i m u m Essential M e d i u m ( I m m u n c h e m i e , Berlin, G D R ) . T h e dissociated cells were transferred to a 0.1 ml, glass-bottomed c h a m b e r that was m o u n t e d on the stage of a Z E I S S inverted microscope. After the cells had settled we perfused the c h a m b e r with the following solution (mM) : 140 K-aspartat, 10 E G T A , 1 M g C 1 2 , 5 Hepes, p H 7.4 with K O H . Patch clamp recordings were performed in this medium. T h e solution has the a d v a n t a g e that the cells are completely quiescent a n d the resting potential is near 0 m V [17]. T h e patch clamp electrodes were filled with a Hepes buffered saline solution (raM): 140 NaC1, 5.4 K H E P O 4 , 2.5 CaCI2, 1 MgC12, 11 glucose, 5 Hepes, titrated with N a O H to p H 7.4. All experiments were done at room temperature (20~ T h e patch pipettes were fire polished

Please address all correspondenceto : B. Nilius,at the above address. 0022-2828/87/090865 + 10 $03.00/0

9 1987 Academic Press Limited

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B. N i l i u s et al.

to a final diameter less than 1 # m (resistance more t h a n 10 Mfl). After formation of giga o h m seals we started in the cell attached configuration of patch clamping. N a channels were present in almost every patch. After checking the voltage range of c h a n n e l openings we m a n u a l l y excised the patches a n d c o n t i n u e d in all the experiments in the insideout mode. Lidocaine (Xylocitin, J e n a p h a r m , G D R ) was applied to the b a t h in concentrations between 1 a n d 100 #M. Patch clamp device was s t a n d a r d [15]. We used a n 8 bit A/D conversion with a sampling rate of 10 kHz in most of the experiments. T h e samples were filtered with a n analog 3. order Bessel filter set to 2 kHz. M e a n currents were determined by averaging the current signals at each time point of the " e n s e m b l e " of 76 identical pulses given sequentially. T h e sweeps consisted of 320 points each a n d were corrected for leakage a n d capacity transients by s u m m i n g a n d averaging the traces with no activity (nulls) a n d subtracting the averaged " n u l l " from (a)

0 5pA

;

each trace. T h e transition between closed and open states were d e t e r m i n e d using an i/2threshold detection scheme (i : u n i t a r y c u r r e n t [10]). Closed a n d open time distributions were fit to sum-of-exponentials probability density functions. Best a p p r o x i m a t i o n was achieved using a M a r q u a r d t - L e v e n b e r g algor i t h m [3]. T h e analysis program also produced plots of the distribution of the n u m b e r of openings per sweep. Averaged data are presented in m e a n __+S.E.M. Results

Lidocaine block of cardiac Na channels Figure 1 shows the effect oflidocaine on single N a c h a n n e l currents in a n isolated ventricular cell. T w e n t y m i c r o m o l a r lidocaine were sufficient to abolish the averaged current even at a very negative holding potential of - - 1 2 0 m V w h e n applied to the i n n e r side of the memb r a n e in inside-out patches. I n the same patch 5 #M lidocaine reduced the peak of the aver(b)

(c)

5pM

20gM

J

5 ms

5ms I Cont

FIGURE 1. Effects of lidocaine on single Na channel currents in a cell free inside-out patch from guinea-pig ventricular cells. The step duration is 30.8 ms, the latency to the depolarization from -- 120 to -60 mV is 1.1 ms. Averaged currents were obtained from 76 sweeps. All the records are from the same patch. (a): control: (b): after application of 5 /ZMlidocaine to the inner surface of the membrane : (c) : channel activity after 20 #M lidocaine. Openings were detected in only 5 of 76 sweeps (cell090786-5).

Single S o d i u m C h ~ n e l s

aged current by about 50% of the control value. These concentrations are considerably smaller than the concentrations used in similar pulse protocols by the application from outside (Bean et al. 1983, Fig. 1). T h e reduction of the averaged current was clearly due to an increased n u m b e r of nulls and an apparent shortening of the channel's openings. T h e single channel unitary current was not significantly affected. F r o m long well resolvable openings the following values could be measured: i = 1.38 4- 0.23 p A at --60 m V (control, n = 5), i = 1.27 + 0.17 pA at --60 m V (5/~M lidocaine, n = 5). T h e small but non-significant decrease in lidocaine treated patches we discuss as a result of bandwidth limitations due to a decreased open time of the channels. L o n g lasting or multiple reopenings of the channel as seen in the left part of Figure 1 were completely prevented by lidocaine (see also Fig. 7). From eight cells the effect of lidocaine on the maximal probability P of the channel to be open and on the relative n u m b e r of nulls was measured. We defined P by P = Im.x/N'i

(I)

and normalized it. Im,x is the peak of the averaged current, i the unitary single channel N a current. The n u m b e r of channels was estimated from the n u m b e r of simultaneously open channels. Figure 2 shows that lidocaine from inside is a very potent blocker of N a channels even at a

negative holding potential. In parallel with the decrease in P the relative n u m b e r of nonempty sweeps (1 -- nulls/sweeps) was decreased. Therefore, the block mainly results from an increased n u m b e r of nulls, e.g. the channel seems to be stabilized in a nonavailable mode. T h e dependence P on the lidocaine concentration was described by P = 1/(1 + [lido]//fm)

te P 9

_ nulls , (I ~ )

0

DidO] )--I

P: ( I + - - ~ -m- . Km=2.9 ~M 0.5-

0

I

w

I0 Lidocaine [/xM]

.

-

I00

F I G U R E 2. Concentration-effect relationship for lidocaine on single Na channel currents. P has been measured from the peaks of the averaged currents and normalized. O : P calculated from equation (1), O : relative number of non-empty sweeps. The smooth line was fitted by equations (2). The points are from eight patches.

(2)

T h e half-blocking concentration for lidocaine acting on the inner side of the membrane was 2.9/~M. This concentration is considerably smaller than described by application from outside ([1, 31], see discussion).

Lidocaine affects the gating behaviour of Na channels We found that lidocaine affected also the gating behaviour of N a channels by reducing the mean time the channel spends in the open configuration. T h e effects of lidocaine on the life time of N a channels were only studied for steps from -- 120 to --60 mV. Figure 3 shows that the untreated channels rarely show longlasting openings and multiple reopenings. F r o m the same patch as shown in Figure 3 we measured in 14 non-overlapping openings in one sweep an averaged open time (7~ of 0.7 and an averaged closed time (7c) of 1.6 ms. T h e likelihood (L) that a train of nonoverlapping events m a y arise from sequential openings of at least two channels was estimated from L(2) =

I

867

(

1+

N~I

9

7~) -14

=

0.7"*

This value predicts that the likelihood is bigger than 99% that sequential occurring non-overlapping openings result from the repetitive activity of one channel [9]. Five micromolar lidocaine reduced not only the n u m b e r of n o n - e m p t y sweeps but also obviously shortened the channel openings. From the exponentially fitted distribution of the open times (same patch as in Fig. 3) a mean open time of 0.32 ms was measured before application of lidocaine. Lidocaine decreased the mean open time to 0.12 ms (Fig. 4). Controls from nine cells revealed a mean open time of 0.44 + 0.17 ms. Five micromolar lidocaine reduced the mean open time to 0.19 4- 0.13 ms (4 cells, see also Fig. 9).

868

B, Nillus

et al. (b)

Col O'41pA

..

9 ~

,J ,

-60

J --120 mV

J

Cont

30ms 5 ~1~ l i d o c a i n e

F I G U R E 3. Effect of 5 #u lidocaine applied from inside to a cell free patch. (a) : control, 30 ms step from -- 120 to --60 mV. (b): channel activity after 5 # u lidocaine. Note the a p p e a r a n c e of very short openings. The averaged currents were obtained from 76 sweeps ( 10 consecutive sweeps, cell 080786-2).

I00

~00

Cont

-I

5/ZM lidocaine

"1 r = 0.32

ms

-r =O. 12 ms

- i

I

-I

- 6 0 mV

_j -,207 0

2

-

4

O

O

2

4

%,.. [ms] F I G U R E 4. Effects of lidocaine on the m e a n open t i m e for the p a t c h s h o w n in Figure 3. U n d e r control conditions a m e a n open time of 0.32 ms has been measured from m o n o - e x p o n e n t i a l fits of the open t i m e distribution. L i d o c a i n e reduced the m e a n open time to 0.,12 ms.

Single Sodium Channels

As first proposed by [24, 25] Na channels might function in different modes each with a unique' rate of inactivation. One mode of gating is characterized by a fast transition into an absorbing state. A second mode allows multiple reopenings causing slow decaying macroscopic currents [23, 24]. Figure 5 shows this type of Na channel gating in 11 consecutive sweeps in a multichannel inside-out patch. Normally channel openings cluster at the very beginning of the depolarizing step or

s 0,:5 pA '~

869

nulls are observed. In a few cases (between 1 and 9% of the sweeps) multiple reopenings can be observed (Fig. 5, sweep 10, 11). This type of openings may account for a slow or ultraslow decaying current through Na channels [24]. Such slow TTX-sensitive currents have already seen in cardiac preparations [4-7, 11] and can also be described in cell free patches. The contribution of the slow decaying current to the peak Na current nicely fits the probability of the occurrence of "burst-

n=76

sweep 5 " ~ " ~

'l

4pA 50 ms -,o

v

J - 8 0 mV F I G U R E 5. Eleven consecutive sweeps from an excised inside out patch to show the occurrence of burst-like openings of Na channels. 2 of 76 sweeps showed long lasting bursts. The conventional gating behaviour is the clustering of short openings at the very beginning of the depolarizing pulse, step from - 8 0 to - 4 0 mV, multi-channel patch, sweep 9 and 12 are nulls, cell 230986-11).

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like" openings [23]. To check the effects of lidocaine on burst-like openings two different approaches have been chosen. (i) Aconitine has been shown to increase the probability of burst like openings [23]. Figure 6 shows an example. T h e control is characterized by one burst out of 76 sweeps. In the presence of Aconitine within the pipette (10 #M) burst-like openings occurred in 75% of the sweeps. Application oflidocaine to the same patch (inside out patches) completely suppressed the bursts whereas the peak current is only decreased by about 40%. (ii) Reopenings can easily be described in the distribution of the n u m b e r of openings per sweeps. T h e control in Figure 7 shows a second h u m p indicating seven sweeps with more than l0 openings [Fig. 7(b)]. Correlated with the occurrence of multiple openings a clear cut slowly decaying Na current can be detected in the semi-log plot of the averaged current [Fig. 7(a)]. After application of lidocaine to the inner surface of the same insideout patch (5 #M), the peak current is decreased but the second h u m p in the distribution of openings and the slow decaying

Control

0.3 'pAt:''

J

- 90 ~v

phase of the averaged current completely disappeared.

Voltage dependence of lidocaine block It is well known that the local anaesthetics block of Na channels depends on m e m b r a n e potential: depolarization increases, hyperpolarization reduces the block [1, 20, 31]. T h e reduction of P in formula (1) was measured at different holding potentials. A 40 m V shift in the holding potential towards negative potentials diminished the blocking effect of 5 #M lidocaine from 74% (Fig. 8, A1, B1) to 11% (Fig. 8, A2, B2). I n three patches a hyperpolarization of 40 m V reduced the blocking effect of 5 #M lidocaine by 63 • 17%. A similar voltage dependence of lidocaine block could be observed for the mean open time (Fig. 9). In Figure 9(a), (b) and (c) the results from the same patch are shown: lidocaine shortened the mean open time from 0.33 ms to 0.16 (holding potential - 120 mV) or 0.32 ms (holding potential --160 mV), respectively. F r o m nine patches, a mean open time of 0 . 4 4 _ 0.17 ms could be measured

Aconitine

Lidocoine

n:76

,-4._ ....

1

J

I

J

;-;; .......... ,, ........

l

FIGURE 6. Lidocaine suppresses long lasting bursts of Na channel openings. Left: control (cell 230986-12), middle: 10 #M aconitine in the patch pipette (cell 230986-17), right: application of 5 ,uMlidocaine to the same patch. The long lasting bursts are completely suppressed. The reduction of the peak current is about 40%. (40 ms step from - 90 to -40 mV, sampling frequency 1 kHz, filter 0.5 kHz, in all other examples the sampling is 10 kHz, 2 kHz filter).

Single Sodium Channels

871

/

(b) I t

(a)

Cont

.

Cont

i

30

E

3 I

30

o

(c)

30

Lido

4

IO

(d)

Lido

0.1

0.01

0

3

0

30

~

4

I

I I0 Openings/sweep

F I G U R E 7. Lidocaine blocks a slowly decaying Na current. From an at least 3 channel patch the averaged current (inset, from 76 sweeps) is a semi-log plotted (O). The control current clearly shows a second slow decaying phase (fast time constant of decay was 2.3 ms, slow time constant 13.8 ms, the contribution of the slow current to the total current was 9%, Figure 7(a), smoothed mean currents were approximated). Figure 7(b) shows the number of openings from the same patch. A second h u m p indicate seven sweeps with more than 10 openings out of 76 sweeps. Figure 7(c) shows the effect of 5/~M lidocaine. T h e slow decay is completely suppressed. T h e fast time constant was calculated to be 2.0 ms. In the 'openings per sweep' plot the second h u m p also disappeared [Fig. 7(d)]. In contrast to the total block of the slowly decaying phase the effect on the peak current is only 42% (cell 090786-2).

AI

e ~

A2

Cont

0

Coni - 60

J

- 120 mV

5 ~1~ lidocaine

--60

BI

A

0.5 B2

-160 mV

5 ms

I

I

I

-16o

-14o

-12o

v. (my) F I G U R E 8. Voltage-dependence of the lidocaine blocks. T h e averaged mean current (AI, A2) is only modestly increased due to hyperpolarization from --120 to - 1 6 0 m V (test step to - 6 0 mV). 5 #~ lidocaine blocks the peak averaged current by 74% at - 1 2 0 m V holding (A1, A2, B1, B2). At - 1 6 0 m V holding potential the block was reduced to 12% [Fig. 8(c)] (cell 090786-1).

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et al.

I00,

(d)

-60 -120 I 0

I00 i

,

,--

I

0 I00

- 1 2 0 mV

7-

..

I

5 HM

( b)

0.5

0,4

0.3

(c) 5 p-M T= 0.:52 ms

0.2

0.1

0

0

2

-

-

4

topen (ms)

0

Cont -120

lidocame -120

-160

mV

FIGURE 9. Relief of lidocaine block by hyperpolarization. (a): control, open time histograms, % = 0.33 ms; (b) open time histogram after application oflidocaine. The mean open time is reduced to 0.16 ms, (c) : exposed to the same concentration oflidocaine the mean open time is increased due to hyperpolarization of the patch (Fig. 9(a), (b), (c) are from the same patch, holding potentials are indicated, test step to - 6 0 mV, cell 220786-3); (d): mean values of the mean open times in excised patches for steps from - 120 or - 160 to - 6 0 mV (inset). Control: 0.44 • 0.17 ms (n = 9), - 120 mV holding potential, 5 #M lidocaine: 0.19 _ 0.13 ms (n = 4), -- 160 mV holding potential, 5 ,UMlidocaine: 0.38 _ 0.02 ms (n = 2).

b e f o r e the a p p l i c a t i o n of l i d o c a i n e . L i d o c a i n e (5 /~M) d e c r e a s e d the m e a n o p e n t i m e of N a channels to 0.19 + 0.13 ms (n = 4) at - 1 2 0 m V h o l d i n g p o t e n t i a l 0.38 _ 0.02 ms (n = 2) at - - 1 6 0 m V h o l d i n g p o t e n t i a l , r e s p e c t i v e l y [Fig. 9(d)'].

Discussion

T h e m a i n o b j e c t i v e of o u r s t u d y was to e v a l u ate the m e c h a n i s m s o f l i d o c a i n e b l o c k on single c a r d i a c N a c h a n n e l s . U p to n o w , o n l y R e u t e r et al. [27] r e p o r t e d a d e c r e a s e in the p r o b a b i l i t y P of N a c h a n n e l s to o p e n d u e to l i d o c a i n e w i t h o u t a f f e c t i n g the single c h a n n e l c o n d u c t a n c e . T h e m a i n findings in o u r s t u d y w e r e : (i) l i d o c a i n e r e d u c e d the a v e r a g e d single c h a n n e l c u r r e n t s m a i n l y by an i n c r e a s e

of the n u m b e r of nulls w i t h o u t c h a n g e s of the size of the u n i t a r y c u r r e n t s ; (ii) l i d o c a i n e decreases t h e m e a n o p e n t i m e of N a c h a n n e l s ; (iii) l i d o c a i n e sensitively blocks l o n g - l a s t i n g bursts o f c h a n n e l o p e n i n g s ; (iv) if used f r o m inside h a l f m a x i m u m effects of l i d o c a i n e w e r e o b s e r v e d at 2.9 /~M w h i c h is c o n s i d e r a b l y s m a l l e r t h a n in m u l t i c e l l u l a r p r e p a r a t i o n s w h e n a p p l i e d f r o m o u t s i d e ; (v) h y p e r p o l a r i z a t i o n o f inside o u t p a t c h e s e x e r t e d a relief f r o m l i d o c a i n e block (Fig. 9) in a g r e e m e n t with cell-attached patches containing 5-10 c h a n n e l s [31]. L i d o c a i n e reduces a v e r a g e d N a c h a n n e l c u r r e n t s m a i n l y by a n increase in the n u m b e r of nulls. T h e s e effects are in a g r e e m e n t w i t h m e a s u r e m e n t s of m a c r o s c o p i c N a c u r r e n t s in o t h e r c a r d i a c tissues [I, 21, 28]. I n o u r e x p e r i m e n t s l i d o c a i n e was a p p l i e d at the i n n e r

Single S o d i u m Channels surface of the m e m b r a n e in inside-out patches. T a k i n g into account the simple voltage protocol o f o u r single c h a n n e l analysis we have to consider the following situation: (i) 30 ms voltage steps were a p p l i e d in a frequency of 1 Hz. W e therefore c a n n o t separate between tonic a n d u s e - d e p e n d e n t block; (ii) the a p p a r ent b i n d i n g constant of lidocaine to the N a channel strongly d e p e n d s on the d i s t r i b u t i o n of channels between resting and i n a c t i v a t e d states (comprising fractions h a n d 1 - h, respectively, [1]) which were not c o m p l e t e l y m e a s u r e d in this study. I f we regard h values between 0.95 and 0.5 we can calculate the range of the a p p a r e n t b i n d i n g constant K m for our conditions. Bean et al. [1] measured the b i n d i n g constant of lidocaine to the resting channel K R near 400 ~uM, to the i n a c t i v a t e d channels K l n e a r 10#M. T h e a p p a r e n t b i n d i n g constant K m can be described by 1

h

K,, - K ,

1--h + - K,

(3)

([1], e q u a t i o n (1)). T h e calculated values for h between 0.95 a n d 0.5 are between 136 a n d 20 #M. I t means the a p p a r e n t b i n d i n g constant of 2 . 9 / ~ found in o u r experiments is between 7 a n d 50 times smaller than in multifibre experiments when lidocaine is a p p l i e d from outside. S t a r m e r et al. [30] p r o p o s e d that the a p p a r ent dissociation constant for local anaesthetics in h e a r t muscle varies with both the m e m b r a n e p o t e n t i a l a n d the fraction of channels with their m 3 gate open. T h e y included a b i n d i n g site for lidocaine with a restricted access due to the closed configuration of the m gate ( " g a t e d restriction to the b i n d i n g site access"). T h e a p p a r e n t b i n d i n g constant in the case of a restricted access was near 350 #M at - 120 mV. I f we suppose a free access to the b i n d i n g site the m e a s u r e d K m value of 2.9/~M for a h o l d i n g potential of - - 1 2 0 m V closely fits a b i n d i n g of lidocaine at a site with a true K D value o f 10 nM at 0 m V (see e q u a t i o n (17) and Fig. 2 in 30). It can be supposed t h a t in inside-out patches with lidocaine a p p l i e d from the inner surface the b i n d i n g site is not g u a r d e d b y the closed m g a t e conformation. I n this sense it seems to be w o r t h m e n t i o n i n g t h a t lidocaine acts from inside in n e a r l y the same sensitivity as from outside T T X [27, 31].

873

T h e described p H d e p e n d e n c e of lidocaine action as elsewhere p o i n t e d o u t [12, 14, 18, 22] cannot a c c o u n t for the smaller concentrations of lidocaine used in our experiments at a p H o f 7.4 in the inside a n d outside solution. It was obvious that lidocaine strikingly c h a n g e d the g a t i n g b e h a v i o u r of N a channels. T h e finding that the d r u g decreases the m e a n open time of c a r d i a c N a channels m i g h t be i n t e r p r e t e d as a direct action of lidocaine on the open conformation of the channel protein. T a k i n g into account a filtering of 2 kHz the true m e a n open time o f l i d o c a i n e modified N a channel should be even shorter than it is reflected by our values. Also the n u m b e r of nulls seems to be o v e r e s t i m a t e d in our a p p r o a c h because of n o n c o r r e c t i n g our d a t a for missed events. W e showed that lidocaine strikingly influenced the m u l t i p l e reopenings of N a channels. It is well k n o w n t h a t a slow d e c a y i n g or n o n - i n a c t i v a t e d (window) N a current is effected b y lidocaine [5, 6, 8]. L i d o c a i n e can p r o b a b l y block a fraction o f activated N a channels t h a t do only very slowly inactivate a n d can therefore shorten the action potential d u r a t i o n [1, 6, 8]. A n i n t r i g u i n g c a n d i d a t e for these effects is the state of the N a channel showing a high p r o b a b i l i t y of reopenings ([23, 24], for skeletal muscle see also [25, 26]). I n our experiments between one a n d m a x i m a l 9% of the sweeps showed m u l t i p l e reopenings. Colatsky [8] described a w i n d o w - c u r r e n t of a b o u t 0 . 2 / z A / c m 2 a n d c a l c u l a t e d a ratio of the steady state to p e a k N a current to be a b o u t 0.1%. G i n t a n t et al. [11] measured slow T T X - s e n s i t i v e N a currents being a b o u t 0.1 to 1% of the p e a k N a current. I n the experiments of C a r m e l i e t [5] the slow N a current represents between 1 a n d 3% of the fast current which closely m a t c h e s the contribution of sweeps with burst-like openings in single channel recordings. W e found that bursting activity could never be observed in patches t r e a t e d with lidocaine as low as 5 #~. Even in cases where the p r o b a b i l i t y of the appearance of multiple reopenings is increased lidocaine suppressed completely both the bursts a n d the slowly inactivated current (Fig. 6) when still a b o u t 40% of the peak N a c u r r e n t persisted. These findings could refer to an increased sensitivity of the burst-like openings to lidocaine.

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