Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Nav1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC50 ‫؍‬ 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Nav1.8 (IC50 ‫؍‬ 8 nM) and was >100-fold selective vs. human Nav1.2, Nav1.3, Nav1.5, and Nav1.

We report here several unusual features of inactivation of the rat Kv2.1 delayed rectifier potassium channel, expressed in Xenopus oocytes. The voltage dependence of inactivation was U-shaped, with maximum inactivation near 0 mV. During a maintained depolarization, development of inactivation was slow and only weakly voltage dependent (tau = 4 s at 0 mV; tau = 7 s at +80 mV). However, recovery from inactivation was strongly voltage dependent (e-fold for 20 mV) and could be rapid (tau = 0.27 s at -140 mV). Kv2.1 showed cumulative inactivation, where inactivation built up during a train of brief depolarizations. A single maintained depolarization produced more steady-state inactivation than a train of pulses, but there could actually be more inactivation with the repeated pulses during the first few seconds. We term this phenomenon "excessive cumulative inactivation." These results can be explained by an allosteric model, in which inactivation is favored by activation of voltage sensors, but the open state of the channel is resistant to inactivation.

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Antisense (AS) oligodeoxynucleotides (ODNs) targeting the Nav 1.8 sodium channel have been reported to decrease inflammatory hyperalgesia and L5/L6 spinal nerve ligation-induced mechanical allodynia in rats. The present studies were conducted to further characterize Nav 1.8 AS antinociceptive profile in rats to better understand the role of Nav 1.8 in different pain states. Consistent with earlier reports, chronic intrathecal Nav 1.8 AS, but not mismatch (MM), ODN decreased TTX-resistant sodium current density (by 60.5+/-10.2% relative to MM; p<0.05) in neurons from L4 to L5 dorsal root ganglia and significantly attenuated mechanical allodynia following intraplantar complete Freund's adjuvant. In addition, 10 days following chronic constriction injury of the sciatic nerve, Nav 1.8 AS, but not MM, ODN also attenuated mechanical allodynia (54.3+/-8.2% effect, p<0.05 vs. MM) 2 days after initiation of ODN treatment. The anti-allodynic effects remained for the duration of the AS treatment, and CCI rats returned to an allodynic state 4 days after discontinuing AS. In contrast, Nav 1.8 AS ODN failed to reduce mechanical allodynia in the vincristine chemotherapy-induced neuropathic pain model or a skin-incision model of post-operative pain. Finally, Nav 1.8 AS, but not MM, ODN treatment produced a small but significant attenuation of acute noxious mechanical sensitivity in naïve animals (17.6+/-6.2% effect, p<0.05 vs. MM). These data demonstrate a greater involvement of Nav 1.8 in frank nerve injury and inflammatory pain as compared to acute, post-operative or chemotherapy-induced neuropathic pain states.

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, a-dendrotoxin (a-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K + currents of the sensory neurons. a-DTX blocked current in both A-and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to _50 mV and little inactivation during 250 ms pulses. In current-clamp experiments a-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A-and C-type neurons. However, there were prominent effects on excitability. a-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A-and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K + currents function in response to the loss of the a-DTX-sensitive current to effect these changes in action potential wave shape and discharge.

Background-Patients with chronic iron overload may develop a cardiomyopathy manifested by ventricular arrhythmias and heart failure. We hypothesized that iron-loaded cardiomyocytes may have abnormal excitability. Methods and Results-We examined a new model of human iron overload, the Mongolian gerbil given repeated injections of iron dextran. In ventricular myocytes, we measured iron concentration and distribution, action potential, sodium and potassium currents, and sodium channel protein. We showed for the first time that (1) the iron content of gerbil ventricular cardiomyocytes was increased to amounts similar to those of patients with iron-induced cardiomyopathy; (2) the overshoot and duration of the cardiac action potential decreased; (3) sodium current was reduced, steady-state inactivation was enhanced, and single-channel currents were unchanged; and (4) transient outward potassium current was increased, but inwardly rectifying potassium current was unchanged. Neonatal rat cardiomyocytes incubated with iron for 1 to 3 days showed similar changes, and levels of cardiac sodium channel proteins were unchanged. Conclusions-Abnormal excitability and heterogeneous cardiac iron deposition may cause the arrhythmogenesis of human siderotic heart disease. (Circulation. 1999;100:675-683.)

Multiple P2 receptor-mediated mechanisms exist by which ATP can alter nociceptive sensitivity following tissue injury. Evidence from a variety of experimental strategies, including genetic disruption studies and the development of selective antagonists, has indicated that the activation of P2X receptor subtypes, including P2X 3 , P2X 2/3 , P2X 4 and P2X 7 , and P2Y (e.g., P2Y 2 ) receptors, can modulate pain. For example, administration of a selective P2X 3 antagonist, A-317491, has been shown to effectively block both hyperalgesia and allodynia in different animal models of pathological pain. Intrathecally delivered antisense oligonucleotides targeting P2X 4 receptors decrease tactile allodynia following nerve injury. Selective antagonists for the P2X 7 receptor also reduce sensitization in animal models of inflammatory and neuropathic pain, providing evidence that purinergic glial-neural interactions are important modulators of noxious sensory neurotransmission. Furthermore, activation of P2Y 2 receptors leads to sensitization of polymodal transient receptor potential-1 receptors. Thus, ATP acting at multiple purinergic receptors, either directly on neurons (e.g., P2X 3 , P2X 2/3 , and P2Y receptors) or indirectly through neural-glial cell interactions (P2X 4 and P2X 7 receptors), alters nociceptive sensitivity. The development of selective antagonists for some of these P2 receptors has greatly aided investigations into the nociceptive role of ATP. This perspective highlights some of the recent advances to identify selective P2 receptor ligands, which has enhanced the investigation of ATP-related modulation of pain sensitivity.Pain is a multidimensional sensory process that, acutely, is physiologically adaptive in response to dangerous (e.g., sharp, hot, or chemical stimuli) stimuli in the environment. Persistent pain can range from increased sensitivity to mildly painful stimuli (hyperalgesia) or to otherwise innocuous stimuli (allodynia) (Honore and Jarvis, 2006). It is well appreciated that distinct sensory mechanisms contribute to physiological pain, to pain arising from tissue damage (inflammatory or nociceptive pain), and to pain arising from injury to the nervous system (neuropathic pain). Nociceptive pain is caused by the ongoing activation of A-␦ and C-nociceptors in response to a noxious stimulus. It can be further classified into visceral pain, superficial somatic pain, and deep somatic pain (Honore and Jarvis, 2006;Perl, 2007).Tissue injury results in the release of pronociceptive mediators that sensitize peripheral nerve terminals that can ultimately lead to increased excitability of spinal cord dorsal horn neurons. As such, injury-induced sensitization of peripheral nerves facilitates a sensitization of the central nervous system. A multitude of receptors, transmitters, second messenger systems, transcription factors, and other signaling molecules are now appreciated to be involved in pain pathways (Honore and Jarvis, 2006;Perl, 2007).The ability of ATP (Fig. 1) to modulate neural functio...

The structure of the carboxyl half of the pore-forming region of Kv2.1 was studied by replacing each of 15 consecutive residues between positions 383 and 369 with a reporter cysteine residue. Extracellular application of charged, membrane-impermeant methanethiosulfonates irreversibly modified currents at four cysteine-substituted positions, K382, Y380, I379, and D378. Intracellular exposure to methanethiosulfonate ethyltrimethylammonium revealed another set of reactive mutants (V374, T373, T372, and T370). Our results indicate that positions 378 and 374 are exposed at outer and inner mouths of the channel, respectively, and immersed in the aqueous phase. In contrast to present topological models, the 383-369 region appears to span the pore mainly as a nonperiodic structure.