Voltage- and use-dependent inhibition of Na⁺ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent

Abstract

1. Whole cell patch clamp techniques were used to study the effects of 4030W92 (2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine), a new antihyperalgesic agent, on rat dorsal root ganglion (DRG) neurones.

2. In small diameter, presumably nociceptive DRG neurones under voltage-clamp, 4030W92 (1–100 μM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na⁺ currents (TTX_R). From a holding potential (V_h) of −90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC₅₀ value of approximately 103 μM.

3. The inhibitory effect of 4030W92 on TTX_R was both voltage- and use-dependent. Currents evoked from a V_h of −60 mV were inhibited by 4030W92 with a mean IC₅₀ value of 22 μM, which was 5 fold less than the value obtained at −90 mV. Repeated activation of TTX_R by a train of depolarizing pulses (5 Hz, 20 ms duration) enhanced the inhibitory effects of 4030W92. These data could be explained by a preferential interaction of the drug with inactivation states of the channel. In support of this hypothesis 4030W92 (30 μM) produced a significant hyperpolarizing shift of 10 mV in the slow inactivation curve for TTX_R and markedly slowed the recovery from channel inactivation.

4. Fast TTX-sensitive Na⁺ currents (TTX_S) were also inhibited by 4030W92 in a voltage-dependent manner. The IC₅₀ values obtained from V_hs of −90 mV and −70 mV were 37 μM and 5 μM, respectively. 4030W92 (30 μM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTX_S.

5. High threshold voltage-gated Ca²⁺ currents were only weakly inhibited by 4030W92. The reduction in peak Ca²⁺ current amplitude produced by 100 μM 4030W92 was 20±6% (n=6). Low threshold T-type Ca²⁺ currents were inhibited by 17±8% and 43±3% by concentrations of 4030W92 of 30 μM and 100 μM, respectively (n=6).

6. Under current clamp, some cells exhibited broad TTX-resistant action potentials whilst others showed fast TTX-sensitive action potentials in response to a depolarizing current injection. In most cells a long duration (800 ms) supramaximal current injection evoked a train of action potentials. 4030W92 (10–30 μM) had little effect on the first spike in the train but produced a concentration-related inhibition of the later spikes. The number of spikes per train was significantly reduced from 9.7±1.5 to 4.2±1.0 and 2.6±1.1 in the presence of 10 μM and 30 μM 4030W92, respectively (n=5).

7. Thus, 4030W92 is a potent voltage- and use-dependent inhibitor of Na⁺ channels in sensory neurones. This profile can be explained by a preferential action of the drug on a slow inactivation state of the channel that results in a delayed recovery to the resting state. This state-dependent modulation by 4030W92 of Na⁺ channels that are important in sensory neurone function may underlie or contribute to the antihyperalgesic profile of this compound observed in vivo.

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