Actions of the novel neuroprotective agent, lifarizine (RS‐87476), on voltage‐dependent sodium currents in the neuroblastoma cell line, N1E‐115

Abstract

1 The actions of the neuroprotective agent, lifarizine , on voltage-dependent Na+ currents have been examined in the neuroblastoma cell line, NlE-1 15, using the whole-cell variant of the patch clamp technique. 2 At a holding potential of -80mV, lifarizine reduced the peak Na+ current evoked by a lOms depolarizing step with an IC50 of 1.3pLM. At holding potentials of -100 and -60mV the IC50 concentrations of lifarizine were 7.3pM and 0.3LM, respectively.3 At a holding potential of -100 mV, most channels were in the resting state and the IC50 value for inhibition of Na+ current should correspond to the dissociation constant of lifarizine for resting channels (KR). KR was therefore estimated to be 7.3pLM. 4 In the absence of lifarizine, recovery from inactivation following a 20s depolarization from -100 mV to 0 mV was complete within 2 s. However, in the presence of 311M lifarizine recovery took place in a biexponential fashion with time constants of 7 s and 79 s. 5Lifarizine (1 M) had no effect on steady-state inactivation curves when conditioning pre-pulses of 1 s duration were used. However, when pre-pulse durations of 1 min were used the curves were shifted to the left by lifarizine by about 10 mV. Analysis of the shifts induced by a range of lifarizine concentrations revealed that the apparent affinity of lifarizine for the inactivated state of the channel (K,) was 0.19 JM.6 Lifarizine (1I M) had no effect on chloramine-T-modified Na+ currents, suggesting no significant open channel interaction. 7 Taken together, these data show that lifarizine is a potent voltage-dependent inhibitor of Na+ currents in NIE-115 cells and that the voltage-dependence arises from an interaction of the compound with the inactivated state of the channel. The possible contribution of Na+ current inhibition to the neuroprotective actions of lifarizine is discussed.

[3H]‐lifarizine, a high affinity probe for inactivated sodium channels

“…The most likely explanation for these findings is that lifarizine binds selectively to inactivated sodium channels, since these will predominate both in the membrane preparation and in the depolarized synaptosomal preparation. The lower affinity of [3H]-lifarizine in nondepolarized synaptosomes may be due to a proportion, albeit unknown, of the sodium channels in this preparation being in the resting state, for which lifarizine has very low affinity (McGivern et al, 1995).…”

“…Electrophysiological studies have demonstrated that lifarizine has negligible interaction with the open state of the sodium channel (McGivern et al, 1995). In contrast, [3H]-BTX interacts preferentially with open sodium channels (Catterall, 1980 …”

Neuroprotective properties of lifarizine compared with those of other agents in a mouse model of focal cerebral ischaemia

“…In vitro lifarizine has been shown to allosterically interact with the toxin site 2 on sodium channel (IC50 55 nm, inhibition of [3H]-batrachotoxin binding), protects against the toxicity induced by the sodium channel activator, veratridine in chick myocytes (Patmore et al, 1991), and in primary cultures of cortical neurones (May et al, 1995). Lifarizine also potently blocks sodium currents in NIE-115 neuroblastoma cells (Ki 0.2 uM; McGivern et al, 1995).…”

“…Lifarizine allosterically interacts with the toxin site 2 on the sodium channel (IC50 55 nM, inhibition of batrachotoxin binding) and potently blocks sodium currents in NlE-115 neuroblastoma cells with an IC50 of 1.3 gM at a holding potential of -80 mV. The potency is modulated by changing the holding potential (7 yM at -100 mV; 0.3 pM at -60 mV) and has been shown to interact predominantly with the inactivated state of the channel (McGivern et al, 1995).…”

Inhibition by lifarizine of intracellular Ca2+ rises and glutamate exocytosis in depolarized rat cerebrocortical synaptosomes and cultured neurones

“…Preliminary studies in man have indicated that the drug is generally well tolerated, and some evidence of benefit in stroke patients has been obtained (Brown et al, 1995a). In terms of proposed mechanism, electrophysiology studies have shown that lifarizine inhibits sodium currents in a voltage-dependent manner (Sheridan et al, 1991;McGivern et al, 1995). This property arises predominantly from an interaction of lifarizine with the inactivated state of the sodium channel, and consistent with this it was found that the potency of lifarizine in inhibiting sodium channels, and in the affinity of [3H]-lifarizine binding to sodium channels, both increased at more depolarized holding potentials MacKinnon et al, 1995).…”