Saab Carl Y, Cummins Theodore R, Waxman Stephen G
Department of Neurology and PVA/Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale Medical School, New Haven, CT 06510, USA.
Exp Brain Res. 2003 Oct;152(4):415-9. doi: 10.1007/s00221-003-1565-7. Epub 2003 Jul 24.
Tetrodotoxin-resistant (TTX-R) sodium current in small-size dorsal root ganglia (DRG) neurons is upregulated by prostaglandin E(2) and serotonin through a protein kinase A (PKA)/protein kinase (PKC) pathway, suggesting G protein modulation of one or more TTX-R channels in these neurons. Recently, GTP(gammaS), a hydrolysis-resistant analogue of GTP, was shown to increase the persistent current produced by the TTX-R Na(v)1.9. In this study, we investigated the modulation of another TTX-R channel, Na(v)1.8, by GTP(gammaS) in small-diameter DRG neurons from rats using whole-cell voltage clamp recordings. Because it has been suggested that fluoride, often used in intracellular recording solutions, may bind to trace amounts of aluminum and activate G proteins, we recorded Na(v)1.8 currents with and without intracellular fluoride, and with the addition of deferoxamine, an aluminum chelator, to prevent fluoride-aluminum binding. Our results show that GTP(gammaS) (100 micro M) caused a significant increase in Na(v)1.8 current (67%) with a chloride-based intracellular solution. Although the inclusion of fluoride instead of chloride in the pipette solution increased the Na(v)1.8 current by 177%, GTP(gammaS) further increased Na(v)1.8 current by 67% under these conditions. While the effect of GTP(gammaS) was prevented by pretreatment with H7 (100 micro M), a non-selective PKA/PKC inhibitor, the fluoride-induced increase in Na(v)1.8 current was not sensitive to H7 (100 micro M), or to inclusion of deferoxamine (1 mM) in the intracellular solution. We conclude that G protein activation by GTP(gammaS) increases Na(v)1.8 current through a PKA/PKC mechanism and that addition of fluoride to the pipette solution further enhances the current, but is not a confounding variable in the study of Na(v)1.8 channel modulation by G proteins independent of a PKA/PKC pathway or binding to aluminum.
前列腺素E2和5-羟色胺通过蛋白激酶A（PKA）/蛋白激酶C（PKC）途径上调小尺寸背根神经节（DRG）神经元中的河豚毒素抗性（TTX-R）钠电流，这表明G蛋白对这些神经元中一个或多个TTX-R通道具有调节作用。最近，GTP（γS），一种GTP的抗水解类似物，被证明可增加TTX-R Na(v)1.9产生的持续性电流。在本研究中，我们使用全细胞膜片钳记录法，研究了GTP（γS）对大鼠小直径DRG神经元中另一种TTX-R通道Na(v)1.8的调节作用。因为有人提出，细胞内记录溶液中常用的氟化物可能会与痕量铝结合并激活G蛋白，所以我们记录了有无细胞内氟化物以及添加去铁胺（一种铝螯合剂）以防止氟化物-铝结合时的Na(v)1.8电流。我们的结果表明，在基于氯化物的细胞内溶液中，GTP（γS）（100μM）使Na(v)1.8电流显著增加（67%）。尽管移液管溶液中用氟化物代替氯化物可使Na(v)1.8电流增加177%，但在这些条件下，GTP（γS）可使Na(v)1.8电流进一步增加67%。虽然用非选择性PKA/PKC抑制剂H7（100μM）预处理可阻止GTP（γS）的作用，但氟化物诱导的Na(v)1.8电流增加对H7（100μM）或细胞内溶液中添加去铁胺（1 mM）不敏感。我们得出结论，GTP（γS）激活G蛋白通过PKA/PKC机制增加Na(v)1.8电流，并且向移液管溶液中添加氟化物可进一步增强电流，但在研究G蛋白对Na(v)1.8通道的调节作用时，它不是一个独立于PKA/PKC途径或与铝结合的混杂变量。
