Block of Na Channels in Sensory Neurons

A deeper understanding of the mode of action of the SCBIs required the investigation of their cellular effects on sensory neurons. Abdominal stretch receptors from cockroach adults and M. sexta larvae were shown to be blocked by various pyrazolines

Control 10"5 M RH-5529 Wash

Control 10"5 M RH-5529 Wash

Figure 7 RH-5529 raised the threshold in current-clamped crayfish stretch receptors, without affecting passive membrane resistance. Records show voltage (upper) and current (lower) traces for current steps before, during, and after application of 10mM RH-5529. Corresponding traces superimposed on the right show that the spike threshold was raised by RH-5529, with no change in resting potential or in the response to hyperpolarizing pulses. (Reproduced with permission from Salgado, V.L., 1990. Mode of action of insecticidal dihydropyrazoles: selective block of impulse generation in sensory nerves. Pestic. Sci. 28, 389-411; © Society of the Chemical Industry, permission is granted by John Wiley & Sons Ltd., on behalf of the SCI.)

Figure 7 RH-5529 raised the threshold in current-clamped crayfish stretch receptors, without affecting passive membrane resistance. Records show voltage (upper) and current (lower) traces for current steps before, during, and after application of 10mM RH-5529. Corresponding traces superimposed on the right show that the spike threshold was raised by RH-5529, with no change in resting potential or in the response to hyperpolarizing pulses. (Reproduced with permission from Salgado, V.L., 1990. Mode of action of insecticidal dihydropyrazoles: selective block of impulse generation in sensory nerves. Pestic. Sci. 28, 389-411; © Society of the Chemical Industry, permission is granted by John Wiley & Sons Ltd., on behalf of the SCI.)

(Salgado, 1990), but the slowly adapting stretch receptor (SASR) of the crayfish Procambarus clarkii (Wiersma etal., 1953) was selected for studies of the cellular mechanism of action, because the greater size of its neuron enables intracellular recordings at the site of spike initiation.

Pyrazolines blocked the stretch receptor in crayfish with a potency similar to that in insects. In mechan-oreceptors, membrane deformation resulting from elongation activates stretch-sensitive ion channels that induce a so-called generator current that depolarizes the membrane of the spike initiation zone to the threshold for action potential generation. In other words, the generator current resulting from mechanosensory transduction is encoded as a spike train in the spike initiation zone. Insertion of a microelectrode into the spike-initiation zone of an SASR neuron allowed the function of this trans-duction and spike-encoding process to be studied. Figure 7 shows a SASR neuron studied under current clamp, in which current pulses of varying amplitude were injected to either hyperpolarize or depolarize the membrane. Such currents bypass the sensory transduction process and allow direct assessment of the spike-encoding process. The voltage traces in response to the injected currents are shown in the upper row. For negative or hyperpolarizing pulses, represented as downward deflections of both current and voltage, RH-5529 did not affect the membrane response, indicating that it did not affect passive membrane properties (Figure 7). The singular effect of RH-5529 was to raise the threshold for spike generation in response to depolarizing pulses, making it more difficult for injected currents and, by inference, for generator currents, to elicit spikes. From this result, it was immediately clear that voltage-dependent Na+ channels, whose activation determines the threshold and initiates the action potential, were blocked by the pyrazoline. This was also likely the mechanism of block in the spike initiation zones of CNS pacemaker neurons, where spikes are also generated by the activation of Na+ channels in response to depolarization by summation of synaptic inputs and pacemaker currents.

6.2.4.4. Mechanism of Na+ Channel Block

In order to better understand the action of SCBIs on Na+ channels, further studies were carried out on crayfish giant axons treated with pyrazolines, with techniques that allowed the study of Na+ channels under highly controlled conditions. A first hypothesis to explain the insensitivity of axonal Na+ channels to pyrazolines was that the compounds block Na+ channels in a voltage-dependent manner, and are therefore selective for channels in the spike initiation zone. Voltage-gated Na+ channels are complex proteins, whose function is regulated by membrane potential through voltage-dependent con-formational changes occurring on a timescale from less than a millisecond to several seconds (Pichon and Ashcroft, 1985). At the spike initiation zone, neurons operate near the threshold for action potential generation, in the range of —70 to — 50 mV, where important conformational changes in Na+ channels occur prior to opening. In contrast, axons have a resting potential near —90 mV, where Na+ channels are predominantly in the resting state. However, when axons are depolarized to the range where Na+ channels begin to undergo conformational changes, they become sensitive to pyrazolines (Salgado, 1990). Likewise, the extracellularly recorded compound action potential from motor nerves of M. sexta abdominal ganglia, which was likewise highly insensitive to DCJW, was rendered sensitive by depolarization of the nerves with a high K+ saline (Wing et al., 1998).

Figure 8 shows membrane currents evoked by test pulses to 0 mV before and after equilibration with 10 mM RH-3421 at various holding potentials. For each trace, after initial rapid downward and upward transients, the current became inward and peaked

Figure 8 Dihydropyrazole block appears as a parallel shift of the steady state slow inactivation curve in the direction of hyperpolarization. Ionic current traces were scaled by a common factor so that the peak at -120 mV matched the peak before treatment with the dihydropyrazole. Peak INa was depressed most at depolarized potentials, whereas outward current, IK, was not affected by the treatment. The graph shows plots of peak current normalized to the value at -120 mV. RH-3421 (10mM) appears to shift the steady state inactivation relation to the left by 8.6 mV. (Reproduced with permission from Salgado, V.L., 1992. Slow voltage-dependent block of Na+ channels in crayfish nerve by dihydropyrazole insecticides. Mol. Pharmacol. 41, 120-126; © American Society for Pharmacology and Experimental Therapeutics.)

Figure 9 A model showing the resting (R), Open (O), and inactivated (I) states of the Na+ channel, and specific interaction of the SCBIs (D) with the inactivated state.

Figure 8 Dihydropyrazole block appears as a parallel shift of the steady state slow inactivation curve in the direction of hyperpolarization. Ionic current traces were scaled by a common factor so that the peak at -120 mV matched the peak before treatment with the dihydropyrazole. Peak INa was depressed most at depolarized potentials, whereas outward current, IK, was not affected by the treatment. The graph shows plots of peak current normalized to the value at -120 mV. RH-3421 (10mM) appears to shift the steady state inactivation relation to the left by 8.6 mV. (Reproduced with permission from Salgado, V.L., 1992. Slow voltage-dependent block of Na+ channels in crayfish nerve by dihydropyrazole insecticides. Mol. Pharmacol. 41, 120-126; © American Society for Pharmacology and Experimental Therapeutics.)

within 0.5 ms, then reversed and became outward. The inward (downward) peak is the Na+ current and the outward (upward) steady state current is the K+ current (Pichon and Ashcroft, 1985). Depolarization of the axon to potentials more positive than — 90 mV in the control suppressed the Na+ current by a process known as inactivation, which in this case had a midpoint potential of — 77 mV. Na+ channels have two partially independent inac-tivation processes, known as fast and slow inacti-vation. Fast inactivation occurs on a millisecond timescale and serves to terminate the action potential, while slow inactivation occurs on a much slower timescale, over hundreds of milliseconds, and performs a slow, modulatory function. Slow inactivation occurs at more negative potentials than fast inactiva-tion, and is responsible for the inactivation seen in Figure 8. After equilibration of the axon with RH-3421, there was no effect on either Na+ or K+ current at —120 or at —100mV (Figure 8). At more depolarized potentials, however, the Na+ current was specifically depressed by RH-3421, whereas the K+ current was unaffected. When peak Na+ current is plotted against membrane potential, it appears that

Figure 9 A model showing the resting (R), Open (O), and inactivated (I) states of the Na+ channel, and specific interaction of the SCBIs (D) with the inactivated state.

RH-3421 has shifted the slow inactivation curve by 9 mV to the left.

The next step in the analysis is to consider the mechanism of this apparent shift of slow inactivation. Whereas fast inactivation occurs with a time course of hundreds of microseconds to a few milliseconds, and slow inactivation on the order of tens to hundreds of milliseconds, the changes in peak Na+ current in the presence of SCBIs occur on a much slower timescale, on the order of 15min. This slow readjustment of peak current in response to voltage change can therefore be attributed to a new process: the binding and unbinding of SCBIs to Na+ channels.

The Na+ channel undergoes transitions between many different conformations or states, which can be grouped into resting (R), open (O), and inactivated (I) states, each of which may have several substates. The transitions between these naturally occurring states are shown on the left in Figure 9. In this simplified model, = [R]/[R] + [I], is the steady-state slow inactivation parameter, which depends on membrane potential according to

Peripheral Neuropathy Natural Treatment Options

Peripheral Neuropathy Natural Treatment Options

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