Transient decrease in nociceptor GRK2 expression produces long-term enhancement in inflammatory pain.

In heterozygous mice, attenuation of G-protein-coupled receptor kinase 2 (GRK2) level in nociceptors is associated with enhanced and prolonged inflammatory hyperalgesia. To further elucidate the role of GRK2 in nociceptor function we reversibly decreased GRK2 expression using intrathecal antisense oligodeoxynucleotide (AS-ODN). GRK2 AS-ODN administration led to an enhanced and prolonged hyperalgesia induced by prostaglandin E(2), epinephrine and carrageenan. Moreover, this effect persisted unattenuated 2weeks after the last dose of antisense, well after GRK2 protein recovered, suggesting that transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient activation of protein kinase C epsilon (PKCε), (Aley et al., 2000; Parada et al., 2003b), the enhanced and prolonged hyperalgesia following attenuation of GRK2 is PKCε- and cytoplasmic polyadenylation element binding protein (CPEB)-independent and is protein kinase A (PKA)- and Src tyrosine kinase (Src)-dependent. Finally, rats treated with GRK2 AS-ODN exhibited enhanced and prolonged hyperalgesia induced by direct activation of second messengers, adenyl cyclase, Epac or PKA, suggesting changes downstream of G-protein-coupled receptors. Because inflammation can produce a decrease in GRK2, such a mechanism could help explain a predilection to develop chronic pain, after resolution of acute inflammation.

Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity.

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K(D) 50- and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.

Specific distribution of sodium channels in axons of rat embryo spinal motoneurones.

1. The distribution of Na+ channels and development of excitability were investigated in vitro in purified spinal motoneurones obtained from rat embryos at E14, using electrophysiological, immunocytochemical and autoradiographical methods. 2. One hour after plating the motoneurones (DIV0), only somas were present. They expressed a robust delayed rectifier K+ current (IDR) and a fast-inactivating A-type K+ current (IA). The rapid neuritic outgrowth was paralleled by the emergence of a fast-activating TTX-sensitive sodium current (INa), and by an increase in both K+ currents. 3. The change in the three currents was measured daily, up to DIV8. The large increase in INa observed after DIV2 was accompanied by the onset of excitability. Spontaneous activity was observed as from DIV6. 4. The occurrence of axonal differentiation was confirmed by the fact that (i) only one neurite per motoneurone generated antidromic action potentials; and (ii) 125I-alpha-scorpion toxin binding, a specific marker of Na+ channels, labelled only one neurite and the greatest density was observed in the initial segment. Na+ channels therefore selectively targeted the axon and were absent from the dendrites and somas. 5. The specific distribution of Na+ channels was detectable as soon as the neurites began to grow. When the neuritic outgrowth was blocked by nocodazole, no INa developed. 6. It was concluded that, in spinal embryonic motoneurone in cell culture, Na+ channels, the expression of which starts with neuritic differentiation, are selectively addressed to the axonal process, whereas K+ channels are present in the soma prior to the neuritic outgrowth.