Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB+-positive than that in IB(4)-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amount of current injection to evoke action potentials. 4-AP significantly decreased the latency of firing and increased the firing frequency in IB4-positive but not in IB4-negative neurons. Additionally, IB4-positive neurons are immunoreactive to Kv1.4 but not to Kv1.1 and Kv1.2 subunits. Collectively, this study provides new information that 4-AP-sensitive A-type Kv currents are mainly present in IB4-positive DRG neurons and preferentially dampen the initiation of action potentials of this subpopulation of nociceptors. The difference in the density of A-type Kv currents contributes to the distinct electrophysiological properties of IB4-positive and -negative DRG neurons.

The heterotrimeric G protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors and proteins. GPCRs are widely distributed in the peripheral and central nervous systems and are one of the most important therapeutic targets in pain medicine. GPCRs are present on the plasma membrane of neurons and their terminals along the nociceptive pathways and are closely associated with the modulation of pain transmission. GPCRs that can produce analgesia upon activation include opioid, cannabinoid, α 2 -adrenergic, muscarinic acetylcholine, γ-aminobutyric acid B (GABA B ), group II and III metabotropic glutamate, and somatostatin receptors. Recent studies have led to a better understanding of the role of these GPCRs in the regulation of pain transmission. Here, we review the current knowledge about the cellular and molecular mechanisms that underlie the analgesic actions of GPCR agonists, with a focus on their effects on ion channels expressed on nociceptive sensory neurons and on synaptic transmission at the spinal cord level.

Opioids have a selective effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not fully known. Two broad classes of nociceptors can be distinguished based on their growth factor requirements and binding to isolectin B 4 (IB 4 ). In this study, we determined the difference in the modulation of voltage-gated Ca 2ϩ currents by [D-Ala 2 ,N-Me-Phe 4 ,Gly-ol 5 ]-enkephalin (DAMGO, a specific opioid agonist) between IB 4 -positive and -negative small dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated DRG neurons in adult rats. Both 1-10 M DAMGO and 1 to 10 M morphine had a greater effect on high voltage-activated Ca 2ϩ currents in IB 4 -negative than IB 4 -positive cells. However, DAMGO had no significant effect on T-type Ca 2ϩ currents in both groups. The N-type Ca 2ϩ current was the major subtype of Ca 2ϩ currents inhibited by DAMGO in both IB 4 -positive and -negative neurons. Although DAMGO had no effect on L-type and R-type Ca 2ϩ currents in both groups, it produced a larger inhibition on N-type and P/Q-type Ca 2ϩ currents in IB 4 -negative than IB 4 -positive neurons. Furthermore, double labeling revealed that there was a significantly higher opioid receptor immunoreactivity in IB 4 -negative than IB 4 -positive cells. Thus, these data suggest that N-and P/Qtype Ca 2ϩ currents are more sensitive to inhibition by the opioids in IB 4 -negative than IB 4 -positive DRG neurons. The differential sensitivity of voltage-gated Ca 2ϩ channels to the opioids in subsets of DRG neurons may constitute an important analgesic mechanism of opioids.The dorsal root ganglion (DRG) neurons and the associated primary afferent nerves transmit different sensory modalities, including nociception to the spinal dorsal horn. The DRG neurons are a mixed population of cells that differ in size, phenotype, and central projections in the dorsal horn. The slowly conducting A␦-and C-fiber afferents are connected to small DRG neurons and transmit primarily nociceptive information. Opioids have a distinct effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms of this differential effect are not fully known. One of the important analgesic mechanisms of opioids is through inhibition of synaptic transmission by acting on voltage-gated Ca 2ϩ channels present on the central terminals of DRG neurons (Rusin and Moises, 1995;Kohno et al., 1999). It has been demonstrated that opioids inhibit voltage-gated Ca 2ϩ channels in dissociated DRG neurons from 10 to 90% (Schroeder and McCleskey, 1993;Moises et al., 1994). It is likely that the large difference of the opioid effect on Ca 2ϩ channels is due to the heterogeneity of DRG neurons studied. The opioid sensitivity and voltage-gated Ca 2ϩ channels in different classes of DRG neurons remain unclear.A novel approach to the classification of DRG neurons is derived from the requirements of subsets of neurons for specific...

Here, we present novel evidence that 4-AP and several of its analogs directly stimulate high voltage-activated Ca 2؉ channels (HVACCs) in acutely dissociated neurons. 4-AP, 4-(aminomethyl)pyridine, 4-(methylamino)pyridine, and 4-di(methylamino)pyridine profoundly increased HVACC, but not T-type, currents in dissociated neurons from the rat dorsal root ganglion, superior cervical ganglion, and hippocampus. The widely used Kv channel blockers, including tetraethylammonium, ␣-dendrotoxin, phrixotoxin-2, and BDS-I, did not mimic or alter the effect of 4-AP on HVACCs. In HEK293 cells expressing various combinations of N-type (Cav2.2) channel subunits, 4-AP potentiated Ca 2؉ currents primarily through the intracellular ␤ 3 subunit. In contrast, 4-AP had no effect on Cav3.2 channels expressed in HEK293 cells. Furthermore, blocking Kv channels did not mimic or change the potentiating effects of 4-AP on neurotransmitter release from sensory and motor nerve terminals. Thus, our findings challenge the conventional view that 4-AP facilitates synaptic and neuromuscular transmission by blocking Kv channels. Aminopyridines can directly target presynaptic HVACCs to potentiate neurotransmitter release independent of Kv channels. Aminopyridines such as 4-aminopyridine (4-AP)3 and 3,4-diaminopyridine (3,4-DAP) are widely used as voltage-activated K ϩ (Kv) channel blockers and can improve neuromuscular function in patients with multiple sclerosis (1), spinal cord injury (2), myasthenia gravis (3), or Lambert-Eaton syndrome (4, 5). The classical view is that the beneficial effect of 4-AP results from blocking Kv channels, which leads to increases in the duration of action potentials, Ca 2ϩ influx, and neurotransmitter release (6 -8). This conception assumes that voltageactivated Ca 2ϩ channels (VACCs) are stimulated indirectly by increased excitability of cells after Kv channels are blocked by 4-AP. However, other Kv channel blockers such as tetraethylammonium (TEA) have limited effects in potentiating neurotransmitter release and improving neuromuscular function. Furthermore, 4-AP is effective in the treatment of patients with VACC antagonist overdose (9), Lambert-Eaton syndrome caused by impairment of presynaptic VACCs (10), or episodic ataxia type 2, a disorder caused by mutation of Cav2.1 (11). These observations raise the possibility that 4-AP may directly stimulate VACCs in addition to its effect on Kv channels.Although VACCs are essential for synaptic and neuromuscular transmission, there is no evidence that 4-AP can directly stimulate VACCs in neurons. In this study, we investigated the direct effect of 4-AP on VACCs in dissociated neurons and its role in potentiating neurotransmitter release. We discovered that 4-AP and several of its analogs have a profound potentiating effect on high voltage-activated Ca 2ϩ channels (HVACCs) independent of Kv channels. The intracellular ␤ subunit is largely responsible for this potentiating effect. Furthermore, 4-AP potentiates neurotransmitter release from both sensory and motor nerve te...

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Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.