Voltage- and use-dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent.
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 microM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na+ currents (TTXR). From a holding potential (Vh) of -90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC50 value of approximately 103 microM. 3. The inhibitory effect of 4030W92 on TTX(R) was both voltage- and use-dependent. Currents evoked from a Vh of -60 mV were inhibited by 4030W92 with a mean IC50 value of 22 microM, 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 microM) 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 (TTXs) were also inhibited by 4030W92 in a voltage-dependent manner. The IC50 values obtained from Vhs of -90 mV and -70 mV were 37 microM and 5 microM, respectively. 4030W92 (30 microM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTXs. 5. High threshold voltage-gated Ca2+ currents were only weakly inhibited by 4030W92. The reduction in peak Ca2+ current amplitude produced by 100 microM 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 microM and 100 microM, 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 microM) 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 microM and 30 microM 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.
Neurosciences Unit, Glaxo Wellcome Research and Development, Stevenage, Hertfordshire.
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D000200:Action Potentials / Q000187:drug effects; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008297:Male; D009475:Neurons, Afferent / Q000187:drug effects* / Q000502:physiology; D018408:Patch-Clamp Techniques; D011743:Pyrimidines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D026941:Sodium Channel Blockers; D013779:Tetrodotoxin / Q000494:pharmacology
C114513:2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine; D018712:Analgesics, Non-Narcotic; D015220:Calcium Channels; D011743:Pyrimidines; D026941:Sodium Channel Blockers; D013779:Tetrodotoxin