State-dependent inhibition of Na+ currents by the neuroprotective agent 619C89 in rat hippocampal neurons and in a mammalian cell line expressing rat brain type IIA Na+ channels

~ Pergamon

Neuroscience Vol. 73, No. 4, pp. 951-962, 1996 Copyright ~, 1996 IBRO. Published by ElsevierScience Lid Printed in Great Britain PII: S0306-4522(96)00092-9 0306-4522/96 $15.00 + 0.00

S T A T E - D E P E N D E N T I N H I B I T I O N OF N a + C U R R E N T S BY THE N E U R O P R O T E C T I V E A G E N T 619C89 IN RAT H I P P O C A M P A L N E U R O N S A N D IN A M A M M A L I A N CELL LINE E X P R E S S I N G R A T B R A I N TYPE IIA N a ÷ CHANNELS X. M. X I E * t and J. G A R T H W A I T E , tNeuroscience Research Group, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K. :~The Cruciform Project, University College London, 140 Tottenham Court Road, London WlP 9LN, U.K. Abstract--The compound 619C89 [4-amino-2-(4-methyl- l-piperazinyl)-5-(2,3,5-trichlorophenyl)pyrimidine] is an effective neuroprotective agent in in vivo models of cerebral ischaemia. It has been suggested to act by inhibiting voltage-gated Na ÷ channels. To test this hypothesis, the action of 619C89 on recombinant rat brain type I I A N a ÷ channels expressed in Chinese hamster ovary cells and on native Na ÷ channels in acutely dissociated rat hippocampal neurons has been studied using whole-cell voltage-clamp recording techniques. In the cell line expressing type I I A N a ÷ channels, 619C89 caused a reversible inhibition of Na ÷ currents in a concentration- and voltage-dependent manner. A half-maximal inhibitory concentration 0c50) of approximately 50 tzM was obtained at a holding potential of - 9 0 mV whereas, with a conditioning prepulse to - 6 0 mV for 30 s, the Ics0 was reduced to 8/~ M. Furthermore, the inhibition was markedly enhanced by a use-dependent action, which was dependent not only on the frequency of stimulation, but also on the duration (3.540 ms) of the pulses. Trains (10-50 Hz) of up to 60 depolarizing pulses of 0.7 ms duration did not evoke any use-dependent inhibition in the presence of 619C89, suggesting that this compound is not an open channel blocker. The voltage- and use-dependent inhibition by 619C89 was also observed on native Na ÷ channels in hippocampal neurons. 619C89 (10 ktM) produced a small hyperpolarizing shift in the fast inactivation curve and a substantial (13mV) hyperpolarizing shift in slow inactivation. The compound dramatically delayed the recovery from inactivation without affecting the development of inactivation. Moreover, 619C89 has no effect on the shape of the current-voltage relationship or on the voltage activation curve. These data indicate that 619C89 interacts selectively with the inactivated state of the Na ÷ channel with an estimated affinity of 3 ktM. This primary action of 619C89 may underlie its neuroprotective effects. Copyright ~ 1966 IBRO. Elsevier Science Ltd. Key words: sodium channel inhibitor, anoxia, ischaemia, stroke, whole-cell recording, potassium currents.

Inhibition of voltage-gated Na + channels, thereby preventing further membrane depolarization and the subsequent ionic and metabolic disturbances occurring as a result of cerebral ischaemia, represents a potentially effective way of preventing neuronal death. 8"~2"~5'21"22"34 The novel antiepileptic drug, lamotrigine, acting on voltage-gated N a + channels has been shown to have neuroprotective effects in animal *To whom correspondence should be addressed. Present address: Neuroscience Unit, The Medicines Research Centre, Glaxo Wellcome, Gunnels Wood Road, Stevenage, Herts SGI 2NY, U.K. Abbreviations: CHO, Chinese hamster ovary cells; CNalIA, CHO cell line expressing rat brain type IIA Na + channels; EGTA, ethyleneglycol-bis(/~-aminoethyl ether)-N,N,N',N'-tetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; PIPES, piperazine-N,N'-bis-(2-ethanesulphonic acid); TEA, tetraethylammonium; TTX, tetrodotoxin; 619C89, 4- amino- 2- (4-methyl-l-piperazinyl)-5-(2,3,5-trichlorophenyl)pyrimidine; 1003C87, 5-(2,3,5-trichlorophenyl)2,4-diaminopyrimidine. 951

models of cerebral ischaemia. 18'19'29'39Structural analogues of lamotrigine, such as 619C89 [4-amino-2(4-methyl- l - p i p e r a z i n y l ) - 5 - ( 2 , 3 , 5 - t r i c h l o r o p h e n y l ) pyrimidine] and 1003C87 [5-(2,3,5-trichlorophenyl)2,4-diaminopyrimidine] are more effective neuroprotective agents in both focal and global ischaemia models in uivo. 8'12'13"18'19 619C89 has been demonstrated to reduce total brain infarction volume by more than 60% after permanent middle cerebral artery occlusion in rats and is currently in clinical trials for the indication of acute stroke. 2°'21 619C89 is approximately four times more potent than lamotrigine in inhibiting glutamate release evoked by the Na + channel opener, veratrine, but is inactive against high-K+-induced glutamate release. 11'12 Veratrineevoked uptake of [~4C]guanidine into synaptosomes, presumably through open N a ÷ channels, is also inhibited by 619C89. 25 These studies suggest that 619C89 may act primarily on voltage-gated Na ÷ channels.

952

X . M . Xie and J. Garthwaite

The m a i n functional c o m p o n e n t of the N a ÷ channel is the ~-subunit, a n d four distinct types (I, II, IIA a n d III) have been identified in rat b r a i n by c D N A cloning a n d sequencing (for review see Refs 3 a n d 4). R a t b r a i n type I I A N a ÷ c h a n n e l a - s u b u n i t s stably expressed in Chinese h a m s t e r ovary cells ( C N a I I A - l ) generate t e t r o d o t o x i n (TTX)-sensitive N a ÷ currents with c o m p a r a b l e kinetics to those observed in neurons. 3'27 F u r t h e r m o r e , local anaesthetic, a n t i a r r h y t h mic a n d a n t i c o n v u l s a n t drugs, including lamotrigine, interact with these r e c o m b i n a n t N a + c h a n n e l s in a m a n n e r similar to their action o n native channels. 24'39 The present study was u n d e r t a k e n to investigate the action o f 619C89 on the defined type I I A N a + c h a n n e l a n d o n native N a + c h a n n e l s in acutely dissociated rat h i p p o c a m p a l CA1 p y r a m i d a l n e u r o n s using whole-cell voltage-clamp recording techniques. Preliminary results have been presented in a b s t r a c t form. 37,38

EXPERIMENTAL PROCEDURES

Cell cultures

The CNalIA-1 cell line was derived from a Chinese hamster ovary (CHO-K1; American Type Cultures) and stably transfected with a cDNA encoding the rat brain type IIA Na + channel? 7 The cells were cultured in RPMI medium (Gibco) with 10% fetal calf serum and nonessential amino acids (Sigma). Streptomycin (2 mg/ml) and penicillin (4 mg/ml) were added to the medium to inhibit microbial growth. The ceils were grown on poly-D-lysinecoated glass coverslips in a 5% COz atmosphere at 37°C for one to two days before electrophysiological experimentation. Acute dissociation o f hippocampal neurons

Transverse hippocampal brain slices (500#M thick) were prepared from 10-15-day-old Wistar rats (home-bred) using a Vibratome. The slices were placed in a holding chamber for 1 h prior to dissociation in piperazine-N,N'-bis(2-ethanesulphonic acid) (PIPES) solution containing (raM): NaCI 120, KCI 3, MgC1z 2, glucose 11 and PIPES-Na 86, pH adjusted to 7.4 with NaOH and had an osmolality of 280-300 mOsm/l. The solution was bubbled with 100% 02 . Selected slices were then treated for 10 min at 37°C with 3 mg/ml protease XXIII (Sigma) in the PIPES solution. The slices were then transferred to the PIPES solution containing l mg/ml bovine serum albumin and 1 mg/ml trypsin inhibitor (Sigma). The CAt region was isolated from the slices by micropunching and was triturated through fire-polished Pasteur pipettes in the same medium. The top half of the suspension was then transferred onto a poly-D-lysine-coated glass coverslip placed in the recording chamber. The dissociated neurons adhered to the coverslip within 15-20 rain without any perfusion. Thereafter, the neurons were superfused with extracellular recording solutions (see below) and used for electrophysiological studies within 2 3 h. Whole-cell voltage-clamp recording

The dissociated neurons or cultured cells on coverslips were placed in a recording chamber and superfused with different extracellular solutions depending on the type of currents to be studied. The recording chamber volume was approximately 0.2 ml and the flow rate was 1.5-2 ml/min. For recording isolated Na + currents in hippocampal pyramidal neurons, the extracellular solution contained (mM): NaC1 80, tetraethylammonium chloride (TEA) 60, KC1 4.7, MgCI 2 1.2, CaC12 2, CdC12 0.2, glucose 11 and HEPES 5, pH

adjusted to 7.4 with NaOH, with osmolality ranging from 280 to 300mOsm/l. Patch pipettes were filled with an internal solution containing (mM): CsF 120, Cs-EGTA 10, NaC1 12 and HEPES 10, pH 7.2 adjusted with CsOH and of osmolality approximately 300 mOsm/l. For recording type IIA Na ÷ channels in CNallA cells, the same internal solution was used, but the extracellular solution contained (mM): NaC1 140, KC1 4.7, MgC12 1.2, CaC12 1, glucose 11 and HEPES 5, pH 7.4. For recording outward K ÷ currents in neurons, the extracellular solution contained (mM): NaCI 80, choline chloride 60, KC1 4.7, MgC12 1.2, CaC12 2, CdC12 0.2, glucose 11 and HEPES 5, pH 7.4. TTX (0.2/IM) was added after the whole-cell configuration was obtained. Patch pipettes were filled with an internal solution containing (mM): potassium gluconate 116, NaC1 4, KC1 17.5, K-EGTA 0.2, Hepes 10, Mg-ATP 4, pH 7.2 adjusted with KOH, with osmolality around 300 mOsm/1. Currents were recorded at room temperature under whole-cell voltageclamp 9 using single-electrode voltage-clamp amplifiers (Axopatch-lD) and were filtered at 5 kHz. Patch pipettes were pulled from borosilicate glass capillaries and fire polished. Their resistance was 3-5 Mr] when filled with an internal solution. Compensation circuitry was used to minimize series resistance errors and 80-85% of the series resistance (3-5 Mr2) could usually be compensated. In most cases, Na + currents ranged from - 0 . 8 to - 4 n A ; the voltage drop across the compensated series resistance was < 4 mV. The active components of the currents evoked by voltage steps were corrected for leak currents (except for traces shown in Figs 2 and 7) using standard leak subtraction protocols provided with the pCLAMP6 software. Membrane potentials quoted were not corrected for junction potentials ( 2 ~ m V ) . Na + currents recorded from these cells at a holding potential (Vh) of - 9 0 mV always increased progressively within the first 10-15min of recordings and then stabilized for a further 20~10min. 39 Drugs were applied only when the control currents were stable. Most compounds and agents were obtained from Sigma or BDH. 619C89 was synthesized by the Wellcome Research Laboratories (Beckenham, U.K.). Stock solutions of 619C89 mesylate salt (10 mM) were freshly prepared with distilled water and diluted with the extracellular solution to the desired concentrations. Data were digitized and analysed using pCLAMP6 software (Axon Instruments). All results are presented as the mean + S.E.M. Student's t-test was used for statistical evaluation. For construction of activation curves, Na + conductance (gNa) was calculated from the peak current (INa), according to the following equation: gr~a = I N a / ( V - E r J , where V is the membrane potential during test pulses and ENa the reversal potential of the peak current. Na + conductance was plotted against test pulse potentials and fitted by a Boltzmann function according to the equation: g/gmax = 1/[1 + exp(Vi/2 - V/k], where g is the normalized conductance relative to the maximal values (gmax)at the largest positive test pulses in each condition, VL.2 is the membrane potential at which the half-maximal open probability of channels occurs and k is the slope of the curve. For construction of inactivation curves, the peak current (I) was normalized relative to the maximal value (/max) at --90 mV in control conditions and plotted against conditioning pulse potentials. The curves were fitted by a Boltzmann function according to the equation: I/lma x = 1/[1 + e x p ( V - Vt/2)/k], where V is the membrane potential during prepulses, V~/zthe membrane potential for I/l,,,,~x =0.5 and k the slope. The dose-response curves were obtained by fitting the data according to an independent binding site receptor model of the form: Y/Ym,x = 1 -- [B/(B + Kc)]", where y is the normalized current in the presence of 619C89, Ymax is the normalized maximal current in control, B represents drug concentration, n the Hill coefficient and Kc the apparent dissociation constant for the drug.

Sodium channels and neuroprotection

RESULTS Voltage-dependent inhibition of Na + currents by 619C89 in CNalIA cells Fast transient inward currents ( - 1 to - 4 n A ) were elicited by test pulses that depolarized the cell to around 0 m V (10ms duration, 0.1-0.5 Hz) from a Vh of --90 mV in more than 90% of C N a l I A - 1 cells recorded. The current amplitudes were reduced as the extracellular N a + concentration was decreased and

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Na + currents by 619C89 in CNalIA cells. (A) Experiments were performed on the same cells with switched conditioning prepulses. One test pulse depolarized to 0 mV (10 ms) from a holding potential (Vh) of --90 mV and the other was preceded by a conditioning prepulse to - 6 0 mV for 30 s (Vh = - 6 0 m V ) . Na ÷ current traces were elicited before, during and after the application of 10 and 30/~M of 619C89 and have been superimposed. In this and all subsequent figures, capacitative transients were blanked. (B) Dose-response relationship for 619C89 at two different holding potentials. The experimental protocol was the same as shown in A. The data were normalized with respect to the amplitudes of the currents elicited in the absence of the compound at each holding potential [(O) - 9 0 m V ; (@) - 6 0 mV]. Each point is the mean + S.E.M. of three to eight cells. The smooth curves were obtained by fitting the data according to an independent binding site receptor model. The apparent dissociation constant for 619C89 (Kc) and the Hill coefficient (n) were estimated to be 48.4 4- 8.1 #M and 0.91 + 0.1 (+ fitting error) respectively at a Vh of - 9 0 mV, and 8.3 + 1.2 ttM and 0.85 + 0.1 at a Vh of - 6 0 mV. Since n values were close to I the drug:binding site ratio was assumed to be I. Thus, the approximate 1¢50was estimated to be 50#M at a Vh of - 9 0 m V and 81tM at a Vh of 60 mV. -

they were completely blocked by 0.2 p M TTX, confirming that they were mediated by TTX-sensitive Na ÷ channels. 24'27,39 Bath application of 619C89 ( 0 . 5 - 1 0 0 p M ) caused a concentration- and voltagedependent inhibition of the N a ÷ current (Fig. 1A). To directly examine the voltage dependency, cells were subjected to two protocols that were alternated at l-min intervals. One test pulse depolarized the cell to 0 m V (10ms) from a Vh of - 9 0 without any prepulse and the other was preceded by a conditioning prepulse to - 60 mV for 30 s (referred to as Vh of --60 mV). Figure 1A illustrates typical results from the same cell, showing that 10 and 3 0 p M 619C89 inhibited the Na ÷ current amplitude to 81% and 50% of control amplitude, respectively, at a Vh of - 9 0 mV, while with a prepulse to - 6 0 mV, the current was reduced to 47% and 25% of control, respectively. The onset of inhibition produced by 619C89 was rapid ( ~ l m i n ) and the block was reversible within a few minutes of washout of the c o m p o u n d (n = 26). However, currents recorded at a Vh of --60 mV sometimes progressively ran down with time. Thus, the recovery from drug action obtained at Vh of - 6 0 mV usually appears less complete than at a Vh of --90 mV. The dose-response curves obtained from these two different holding potentials revealed a half-maximal inhibitory concentration (ICs0) of approximately 50/~M at a Vh of - 9 0 m V and 8 / t M at a Vh of - - 6 0 m V (Fig. IB).

Use-dependent action of 619C89 on Na + currents

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In addition to the voltage-dependent inhibition observed at low stimulation frequencies ( < 0.1 Hz), some Na + channel inhibitors also exhibit use dependency which enhances drug action during sustained electrical activity. 16,24,39To determine whether 619C89 possesses this property, trains of depolarizing pulses with variable duration were used. To minimize the voltage-dependent action of the compound, all cells were held at - 9 0 mV. Currents elicited by trains of 20 pulses (10 Hz) with different durations (0.7-40 ms) themselves were well maintained under control conditions. In the presence of 10 p M 6t9C89, however, a significant use-dependent inhibition developed, the extent of which (15-70%) increased as the pulse duration increased (3.5-40ms). However, currents generated by trains of 20450 pulses of short duration (0.7 ms), to minimize inactivation, were not inhibited by the drug. This lack of effectiveness was maintained even with 60 short duration (0.7 ms) pulses delivered at a higher frequency (50 Hz) (data not shown). 619C89 causes a hyperpolarizing shift in Na + current inactivation To characterize the interaction of 619C89 with Na + channels, the basic kinetics of activation and inactivation of the channel were examined. Figure 3 shows the current-voltage ( I - V ) relationship obtained under control conditions and in the presence of 10/~M 619C89. The peak current amplitudes

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Fast and slow inactivation may be two independent processes that can be distinguished by varying the durations of stimulus pulse or preconditioning pulse. 26'39 To examine the effect of 619C89 on the onset rate of fast inactivation, time constants, determined by fitting single exponential functions to the decay phase of current traces elicited by depolarizing test pulses, were plotted against different test potentials. 619C89 ( 1 0 # M ) had no effect on the time course of fast inactivation (n = 6; Fig. 4B). In contrast, 619C89 caused a hyperpolarizing shift in both the fast and slow (steady-state) inactivation of the channel. Fast inactivation curves were obtained using short prepulses of duration 10 ms that preceded the test pulse to 0 mV (for 5 ms) and the slow inactivation curves were constructed using a long prepulse of 60 s duration. Although the half-maximal inactivation voltage (V~/2) of the fast inactivation curve in the presence of 10 p M 619C89 was only shifted by 4 mV to more negative potentials as compared to the control ( - 35.5 _+ 0.8 mV, n = 4), the difference was statistically significant (P < 0.05; Fig. 4C). At the same concentration (10/~M), however, 619C89

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elicited by different depolarizing test pulses were inhibited by 619C89, but the shape of the I - V relationship and the reversal potentials (around + 7 0 m V under the experimental conditions) were unaffected. To analyse the voltage dependence of the N a ÷ channel in more detail, activation curves in the presence and absence of 619C89 were constructed, as shown in Fig. 4A. The peak currents have been normalized for m a x i m a l conductance and expressed as a fraction of open channels. The half-maximal activation voltage (Vl/2) of the curve indicates the probability that 50% of channels will open at the test potential and the slope (k) of the curve suggests the effective valence of channel activation. The values of V,/2 and k were - 2 0 . 1 + 2.5 mV and 6.9 mV/e-fold change in control, and - 2 2 . 3 + 2 . 8 m V and 6 . 8 m V / e - f o l d change in the presence of 10/~M 619C89 (n = 4), indicating that 619C89, at the tested concentrations, had no significant effect on the voltage-dependent activation of the channels.

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Sodium channels and neuroprotection

channel inactivation, was not significantly affected by the c o m p o u n d ( 8 . 9 + 0 . 8 m V / e - f o l d in control; 9.8 + 1.3 mV/e-fold in 619C89).

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action, two sets of cell populations were tested and the rates of development of inactivation obtained from the fifth trials, performed at a similar time after formation of whole-cell recording, were compared. The value of z was 42 + 2 ms in control cells (n = 5), which is not significantly different from 1 0 # M 619C89-treated cells (37 + 2ms, n = 6 Fig. 5A). In contrast to the lack of significant effects on the development of inactivation, the rate of recovery from inactivation was markedly delayed by 619C89. Recovery from inactivation was examined using a 100-ms conditioning pulse to 0 m V , followed by a recovery pulse to - 90 mV for varying durations and then a test pulse (0 mV) to determine the extent of channel repriming. The recovery from inactivation under control conditions was fitted by a single exponential function, revealing.a time constant (z) of 2 ms. In the presence of 619C89 (10/tM), the data were best fitted by a double exponential function with Zl of 3 ms and z 2 of 407ms. This indicates that 619C89 slowed the recovery from inactivation, primarily by affecting the slow component (Fig. 5B). Voltage-dependent inhibition o f native neuronal N a +

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Fig. 5. Effects of 619C89 on the development of inactivation and on recovery from the inactivation. (A) Na ÷ currents were elicited by test pulses to 0 mV (5 ms) preceded by conditioning pulses to - 4 5 mV from the Vh of - 9 0 mv for varying durations (from 1 to 220 ms), as indicated in the inset. The current amplitudes (I) were normalized with respect to their maximal currents (/max) obtained with a 1 ms conditioning pulse in control (O) or in 619C89 (10 #M, O). Data were fitted by the equation I/Im, x = a + bexp(-At/Q, where At is conditioning pulse duration and z the time constant of slow inactivation. The values of z were 42 + 2 ms in control and 37 + 2 ms in the compound (n = 6; P > 0.05). (B) 619C89 slowed recovery from slow inactivation. Na ÷ currents were elicited using a 100-ms conditioning pulse to 0 mV, followed by a recovery pulse to - 90 mV for varying durations and then a test pulse (0 mV, 5 ms), as shown in the inset. The amplitudes of the currents evoked by the test pulses were normalized with respect to the currents elicited by the conditioning pulses in each condition, control (O) or 10/LM 619C89 (O, n = 6), and were plotted as a function of the recovery interval. Data obtained in control conditions were fitted by a single exponential function with a time constant (z) of 2 ms. In 10/LM 619C89, the data were fitted by a double exponential function with z I = 3 ms and T2 = 407 ms.

The fraction of inactivated channels increased with the duration of the conditioning pulse and the relationship could be fitted by a single exponential function. The rate of development of inactivation, indicated by the time constant (z), was often naturally accelerated under control conditions when the fifth trial was compared to the first trial (delivered at one trial per minute). Therefore, to evaluate the drug

F o r comparison with the recombinant type IIA Na + channels, we investigated the action of 619C89 on native N a + channels in acutely dissociated hippocampal pyramidal neurons. U n d e r conditions in which voltage-gated outward K + currents were blocked with external T E A and internal Cs + and inward Ca 2 + currents were blocked with Cd 2 +, pharmacologically-isolated inward N a ÷ currents were elicited by 10-ms depolarizing pulses to - 1 0 m V from a Vh of - 9 0 m V . Bath application of 1 0 a M 619C89 caused an inhibition of N a ÷ currents to 90.2___ 2.9% (n = 10) of the control amplitude. To examine the voltage dependency, experiments were performed using the same protocol as in C N a l I A cells. With switched conditioning prepulses to -60mV for 30s, the current was reduced to 47.3___ 7.6% (n = 4 ) of control amplitude in the presence of 10 # M 619C89 (Fig. 6A). In addition to the N a ÷ channel, neuronal membranes possess abundant voltage-gated K ÷ channels, which usually play an opposite role to N a ÷ channels in the regulation of cell excitability. It was of interest to examine whether 619C89 is able to influence outward K ÷ currents. After blockade of Na ÷ inward currents with T T X (0.2/IM) and using a medium containing high-K + inside the recording pipette, an outward K ÷ current was elicited by 60-ms depolarizing pulses to 30 mV from a Vh of - 90 mV (Fig. 6B). 619C89 (101tM) inhibited the outward current to 72.5 + 4.2% of the control amplitude (n = 10; Fig. 1B). However, it is noteworthy that, unlike the voltage-dependent action on N a + currents, the potency of the c o m p o u n d for inhibition of K ÷ currents was not enhanced at relatively positive membrane

Sodium channels and neuroprotection

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Fig. 6. (A) Voltage-dependent inhibition by 619C89 of native Na ÷ currents in hippocampal neurons. Experiments were performed under conditions that block outward K ÷ currents (extracellular TEA and internal Cs ÷) and inhibit inward Ca 2+ currents (200/~M Cd2+). Recordings were made from the same cells with switched conditioning prepulses every minute. One test pulse depolarized to - 1 0 mV (10 ms) from a Vh of - 9 0 m V and the other was preceded by a conditinging prepulse to - 6 0 m V for 30s (Vh = - 6 0 mV), as shown at the top of each current trace. Na ÷ current traces were elicited under control conditions and in the presence of 10#M 619C89 and have been superimposed. (B) 619C89 inhibited outward K ÷ currents in a voltage-independent manner. In the presence of TTX (0.2/~M) and Cd 2÷ (200/~M) to block inward Na ÷ and Ca 2+ currents, isolated outward K ÷ currents were elicited by depolarizing pulses to 30 mV for 60 ms from two holding membrane potentials, as described in A. K ÷ current traces from the same neuron in the presence and absence of 10#M 619C89 have been superimposed.

potentials. A t a Vh o f - 6 0 m V , K + currents were reduced to 70.2 ___5.3% o f the control (n = 4; Fig. 6B). Use-dependent inhibition o f N a ÷ currents in neurons To examine use dependency, trains o f 20 depolarizing pulses to - 10 mV, with variable d u r a t i o n , were used. Trains (10 Hz) with two different d u r a t i o n s of 3.5 a n d 20 ms caused a progressive inhibition u n d e r control conditions. The current a m p l i t u d e ratios o f pulse 20/pulse 1 were 0.93_+ 0.01 with d u r a t i o n o f 3 . 5 m s a n d 0 . 8 7 + 0 . 0 2 with d u r a t i o n o f 2 0 m s

(Fig. 7A). In the presence of 1 0 # M 619C89, the pulse 20/pulse 1 ratios decreased to 0.89 + 0.01 a n d 0.65 + 0.04, respectively. This indicates t h a t the comp o u n d significantly e n h a n c e s the use-dependent inhibition o f the channel. In contrast, trains of up to 60 pulses with short d u r a t i o n ( 0 . 7 m s ) p r o d u c e d no extra use-dependent drug action c o m p a r e d to the control. T h e ratio of pulse 60/pulse 1 was 0.92 _ 0.07 in control a n d 0.91 + 0.14 in the presence o f 619C89 (Fig. 7A). The effect of varying the d u r a - t i o n of the depolarizing pulse on the use-

958

X . M . Xie and J. Garthwaite

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Duration of pulse (ms) Fig. 7. (A) Use-dependent inhibition by 619C89 of native Na ÷ currents in hippocampal neurons. Currents were elicited by a train of 20 pulses to - 10 mV (10 Hz) with varying dtlrations (0.7, 3.5 and 20 ms) under control conditions and in the presence of 10 # M 619C89. Traces evoked by pulse numbers 1 and 20 were superimposed. Membrane potential was held at - 9 0 m V . Leak currents were not subtracted in this experiment. (B) The effects of pulse duration on use-dependent inhibition of Na ÷ currents in both CNalIA cells and neurons. The data were taken from Figs 2 and 7A. The current amplitudes elicited by pulse number 20 were normalized with respect to the current evoked by the first pulse in each train (pulse 20/pulse 1) and plotted against the duration of the depolarizing pulse under control conditions (IS]) and in the presence of 10/tM 619C89 ( I ) for neurons (n = 7) and for CNalIA cells [(O) control; ( 0 ) 10 # M 619C89, n = 6]. d e p e n d e n t action is s u m m a r i z e d in Fig. 7B. A t a high fre-quency, the longer the d u r a t i o n o f pulses, the greater the degree o f inhibition observed following 619C89 t r e a t m e n t in b o t h C N a I I A cells a n d h i p p o c a m p a l neurons. DISCUSSION

State-dependent inhibition of N a ÷ channels T h e present study d e m o n s t r a t e s a direct effect o f 619C89 o n a defined type o f r e c o m b i n a n t N a +

c h a n n e l a n d o n native n e u r o n a l N a + channels. W e utilized the C N a l I A cell line expressing type I I A -subunits, which is k n o w n to be present d o m i n a n t l y in h i p p o c a m p a l p y r a m i d a l neurons, T M to characterize the interaction o f 619C89 with the different functional states o f the channel. In general, the N a ÷ c h a n n e l exists in at least three states: resting (closed), open a n d inactivated. T h e results o b t a i n e d show t h a t 619C89 inhibits type I I A N a ÷ channels in a strong v o l t a g e - d e p e n d e n t m a n n e r , with a n approximately

Sodium channels and neuroprotection six-fold increase in potency when the membrane potential was briefly held at a less negative value ( - 6 0 mV for 30 s, Ics0 = 8 #M) compared to a Vh of - - 9 0 m V 0c50 = 50#M). Holding the membrane potential at a less negative voltage is known to increase the proportion of Na ÷ channels in the slow inactivated state. 4'26'39 The voltage dependency suggests that 619C89 has a much higher affinity for the inactivated state of the Na ÷ channel compared to the resting state. The voltage dependence of the potency of 619C89 obtained at low stimulation frequencies (<0.5 Hz) was further enhanced by a frequency- or use-dependent action. Stronger inhibition by 619C89 was observed at a high frequency (10 Hz) of stimulation with pulse durations of 3.5-40 ms. However, pulses with short duration of 0.7ms, to allow channel opening whilst minimizing inactivation, did not reveal any use dependency (Figs 2 and 7). These results indicate that the use-dependent inhibition occurred only when inactivation developed and accumulated during repetitive stimuli of long duration, suggesting that 619C89 does not act on the open state per se, in contrast to the local anaesthetics, 2-[2,6-dimethylphenyl)amino]-N,N,N-triethyl-2-oxoethanaminium (QX-314) and N - N - d i e t h y l g l y c y l - N - ( 2 , 6 - d i m e t h y l -

phenyl)glycinamide (GEA968), which have a higher affinity for the open s t a t e Y ° The notion of a selective interaction of 619C89 with the inactivated state was further supported by detailed channel kinetic analyses. There were no changes in the shape of the I - V relationship and no positive shift in the activation curve (Figs 3, 4A), confirming that 619C89 does not interfere with channel opening. In contrast, 619C89 (10 p M ) caused a small (4 mV) but significant hyperpolarizing shift in fast inactivation and a substantial shift (13 mV) in slow, steady-state inactivation without inducing significant changes in the slope of the curves (Fig. 4C, D). These effects seem to result from a profound delay in the recovery from inactivation (Fig. 5B), since there was no change in the onset rate of fast inactivation (Fig. 4B) and no significant acceleration of the development of inactivation (Fig. 5A). The affinity of 619C89 for the inactivated Na + channel (Kx) was estimated at 3 p M according to the following equation: ~,2,39 K I = L/[1 + L / K c / e x p ( A V l / 2 / k ) - 1],

where L is drug concentration, Kc is the apparent dissociation constant observed at a Vh of --90mV, AVI/2 is the shift in the half-inactivation voltage produced by the drug at the test concentration, L, and k is the slope of the steady-state inactivation curve under control conditions. The theoretical value of K~, 3 # M , is approximately 16-fold lower than the apparent dissociation constant obtained at a Vh of -- 90 mV. A concentration of 3/~ M is probably easily

959

achievable in brain tissues following administration of neuroprotective doses (10 mg/kg, i.v .) of 619C89 to rats, where peak total brain tissue concentrations of 619C89 range from 10 to 20 # M (Salmon J., personal communication). The affinity of 619C89 for the inactivated Na ÷ channel is apparently four times higher than lamotrigine (Kl of 12#M), 39 which has been shown to be a less effective neuroprotectant compared with 619C89 in ischaemia models in uivo. 8'12'13'28'29 It is of interest to compare their actions on the same type of Na ÷ channel at comparable concentratrions, i.e. at three to four times their respective K~ values. 619C89 (10 pM) appears to have more pronounced effects on resting channels as manifested by 5-10% inhibition of Na ÷ currents at a Vh of --120mV, at which inactivation is absent. In contrast, Na ÷ currents elicited at this holding potential were unaffected by 5 0 p M lamotrigine. 39 Moreover, 619C89 seems to show less selectivity between the fast and slow inactivation compared with lamotrigine. This was evident in the use-dependent inhibition and in the fast inactivation curve (Figs 2B, 4C). Use-dependent drug action by 619C89 was observed using a train of stimuli with a duration of 3.5ms (10Hz), which caused channel opening and fast inactivation. Also, there was a small but significant negative shift in fast inactivation in the presence of 619C89. However, it was shown previously that lamotrigine produced insignificant changes in these two parameters using the same protocols. 39 Whether this lower selectivity on the fast and slow inactivation compared to lamotrigine could explain the more effective neuroprotection seen with 619C89 s'12'28'29 remains to be determined. The characteristics of voltage- and use-dependent inhibition by 619C89 on recombinant type IIA Na ÷ channels also applied to native Na ÷ channels in hippocampal neurons. 619C89 (10 #M) produced an inhibition of Na ÷ currents in a voltage-dependent manner and the potency was within the range observed with type IIA channels (Figs 1B, 6A). However, the use-dependent inhibition of native Na ÷ channels was greater under control conditions compared with the recombinant Na + channels, while 619C89 had a less profound use-dependent drug action on the native Na ÷ channel (Fig. 7B). These small differences may reflect a number of factors, such as the existence of multiple ~-subunits having differential sensitivity to the drug, the presence of different fl-subunits and different states of protein phosphorylation 14 in native neurons. Effects on voltage-gated outward K + currents

In addition to the inhibition of Na + channels, 619C89 apparently inhibited voltage-gated K ÷ channels in hippocampal neurons. An approximately 30% inhibition of outward K + currents by 10 # M 619C89 was observed at a Vh of --90 or --60 mV. At less

960

X. Xie and J. Garthwaite

negative membrane potentials (e.g. - 6 0 m V ) , the fractions of inactivated channels for both Na ÷ and K ÷ increased. It appears, however, that 619C89 has a higher affinity for the inactivated Na ÷ channel, whilst its potency for inhibiting the inactivated K ÷ channels was voltage-independent (Fig. 6). These results indicate that 619C89, to some extent, selectively inhibits Na ÷ currents over K ÷ currents at less negative membrane potentials. Nevertheless, concomitant partial inhibition of both Na ÷ and K ÷ channels at resting states (e.g. - 9 0 mV) would tend to preserve the ability of the cells to generate action potentials, which depends on the ratio of Na ÷ and K ÷ conductances. This may explain why the compound action potential in an isolated optic nerve preparation was maintained in the presence of 619C89 concentrations which provided protection from anoxia/ischaemia.7 This explanation has been applied to tertiary armine anaesthetics in the same experimental model. 31,35 It is noted that riluzole and phenytoin, two other Na ÷ channel inhibitors and neuroprotectants in animal models of cerebral and white matter ischaemia,6,~° also inhibit outward K ÷ currents. 2'17 However, the means by which their interaction with K ÷ channels may relate to their pharmacological effects in vivo is not clear.

Mechan&ms o f neuroprotection The mechanism underlying neuroprotection by 619C89 or lamotrigine was originally proposed to involve the inhibition of excess glutamate release by an action on voltage-gated Na ÷ channels. T M Although the possibility that additional mechanisms contribute to neuroprotection, for example modulation of neuronal Ca 2+ channels, has not been ruled out, the potency of 619C89 or lamotrigine for inhibiting inactivated Na ÷ channels is correlated well with their potencies for inhibiting veratrine-induced glutamate release and broadly with their effectiveness for neuroprotection in vivo. 8'12'13'28'29'39 619C89 has weak affinity for excitatory amino acid binding sites, ~2 and does not block evoked excitatory postsynaptic potentials mediated either by N-methyl-D-aspartate or non-N-methyl-D-aspartate receptors at concentrations up to 100 # M in rat hippocampal brain slices (Batchelor A. M., personal communication). Thus, 619C89 does not appear to interfere directly with either the release or postsynaptic action of glutamate; instead, its ability to depress pathological release of glutamate is likely to be through an action on the inactivated Na + channels. Moreover, it has recently been demonstrated that 619C89, 1003C87, lamotrigine and other drugs (e.g. phenytoin and carbamazepine) able to block Na ÷ channels are capable of protecting against ischaemic damage in rat optic nerve in vitro, a white matter ischaemia model that is independent of glutamate 673135 excitotoxicity.,, '

Concerning the mechanism of neuroprotection by Na ÷ channel inhibitors, there are several possibilities (for review see Ref. 34). First, anoxia or ischaemia will interfere with the production of ATP and hence disrupt the energy-dependent N a ÷ - K + pumps in the plasma membrane. These events will result in depolarization of the membrane and opening of voltage-gated Na ÷ channels. Secondly, the resulting Na ÷ influx will cause an increase in intracellular Ca 2÷ via a variety of mechanisms, including activation of voltage-gated Ca 2÷ channels and reversal of the Na+-Ca 2+ exchange mechanism.6m'35 Finally, Na +dependent glutamate transport can be reversed and produce excessive glutamate release during ischaemia. 32 Drugs such as 619C89, lamotrigine or phenytoin, able to inhibit Na ÷ channels (perhaps including block of non-inactivating Na ÷ currents33), would thus interrupt the ischaemic cascade at a fiarly early stage and thereby result in neuroprotection. Most of these drugs show the characteristics of voltage- and use-dependent inhibition of the Na ÷ channel, 16'24'34'39reflecting a selective interaction with the inactivated state of the channel. A state-dependent inhibition of Na ÷ channels could explain, at least in part, why these drugs have less neurological side-effects compared to other neuroprotective approaches, such as excitatory amino acid antagonism and neuronal Ca 2+ channel blockade. 2122'3a Interestingly, inhibition and/or downregulation of brain Na + channels has been found to occur naturally in turtles which dive underwater for hours. This regulation may contribute to the ability of these animals to survive under anoxic conditions.23

CONCLUSIONS

The measurement of functional states of Na ÷ channels expressed in CHO cells or existing in native neurons under voltage-clamp experiments allows characterization of drug action. Inhibition of Na ÷ currents by 619C89 appears to be due to a modulation of channel gating rather than an open channel block. A selective stabilization of the inactivated state of the Na ÷ channel could directly counter Na ÷ entry into neurons and thus indirectly decrease depletion of ATP stores, Ca 2÷ influx and excessive release of glutamate. These actions may underlie the neuroprotective action of 619C89. State-dependent modulation of Na + channels provides a mechanism for selective drug action under pathological conditions such as anoxia or ischaemia during stroke or following traumatic brain injury, whilst sparing normal function.

Acknowledgements--We are grateful to Ms L. Morgan, Drs T. Peakman and J. Clare for provision of the CNalIA cell cultures. We thank Drs M. Leach and R. Mulrooney for helpful discussions, and Drs J. Salmon and A. M. Batchelor for sharing unpublished data.

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(1995) A comparative study of the protection of CNS white matter from simulated ischaemia by Na + channel inhibitors. Soc. Neurosci. Abstr. 21, p218. 8. Graham S. H., Chen J., Lan J., Sharp F. R. and Simon R. P. (1993) Neuroprotective effects of a use-dependent blocker of voltage-dependent sodium channels, BW619C89, in rat middle cerebral artery occlusion. J. Pharmae. exp. Ther. 269, 854 859. 9. Hamill O. P., Marty A., Neher E., Sakmann B. and Sigworth F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflfigers Arch. 391, 85-100. 10. Hayakawa T., Hamada Y., Maihara T., Hattori H. and Mikawa H. (1994) Phenytoin reduces neonatal hypoxicischemic brain damage in rats. Life Sci. 54, 387-392. 11. Leach M. J., Baxter M. G. and Critchley M. A. (1991) Neurochemical and behavioural aspects of lamotrigine. Epilepsia 32, $4~8. 12. Leach M. J., Swan J. H., Eisenthal D., Dopson M. and Nobbs M. 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In Pharmacology of Cerebral Ischaemia (cds Krieglsteir~ J. and Oberpichler-Schwenk H.), pp. 551-563, Medpharm Scientific, Stuttgart. 23. Perez-Pinzon M. A., Rosenthal M., Sick T. J., Lutz P. L., Pablo J. and Mash D. (1992) Downregulation of sodium channels during anoxia: a putative survival strategy of turtle brain. Am. J. Physiol. 262, R712-R715. 24. Ragsdale D, S., Scheuer T. and Catterall W. A. (1991) Frequency and voltage-dependent inhibition of type I I A N a + channels, expressed in a mammalian cell line, by local anesthetic, antiarrhythmic, and anticonvulsant drugs. Molec. Pharmac. 40, 756-765. 25. Riddall D. R., Clackers M. and Leach M. J. (1993) Correlation of inhibition of veratrine evoked [14C]guanidine uptake with inhibition of veratrine evoked release of glutamate by lamotrigine and its analogues. Can. J. Neurol. 4, Suppl. S181. 26. Ruben P. C., Starkus J. G. and Rayner M. D. (1992) Steady-state availability of sodium channels. 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35. Waxman S. G., Stys P. K. and Ransom B. R. (1992) Role o f N a + conductance and the N a + ~ a 2+ exchanger in anoxic injury of CNS white matter. In Pharmacology of cerebral ischaemia (eds Krieglstein J. and Oberpichler-Schwenk H.), pp. 13-31. Medpharm Scientific, Stuttgart. 36. Westenbroek R. E., Merrick D. K. and Catterall W. A. (1989) Differential subcellular localization of RI and RII Na ÷ channel sybtypes in central neurons. Neuron 3, 695-704. 37. Xie X. M. and Garthwaite J. (1995) Voltage- and use-dependent inhibition of rat brain sodium channels by the neuroprotective agent 619C89. Soc. Neurosci. Abstr. 21, p218. 38. Xie X. M. and Garthwaite J. (1995) State-dependent block of recombinant rat brain type I I A N a ÷ channels by the neuroprotectant 619C89. Brain Res. Ass. Abstr. 12, p74. 39. Xie X. M., Lancaster B., Peakman T. and Garthwaite J. (1995) Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type I I A N a ÷ channels and with native Na ÷ channels in rat hippocampal neurones. Pfl~gers Arch. 430, 437-446.

(Accepted 14 February 1996)