Choi Jin-Sung, Hudmon Andy, Waxman Stephen G, Dib-Hajj Sulayman D
Department of Neurology, Yale University School of Medicine, New Heaven, CT, USA.
J Neurophysiol. 2006 Jul;96(1):97-108. doi: 10.1152/jn.00854.2005. Epub 2006 Apr 5.
Sodium channel Nav1.8 produces a slowly inactivating, tetrodotoxin-resistant current, characterized by recovery from inactivation with fast and slow components, and contributes a substantial fraction of the current underlying the depolarizing phase of the action potential of dorsal root ganglion (DRG) neurons. Nav1.8 C-terminus carries a conserved calmodulin-binding isoleucine-glutamine (IQ) motif. We show here that calmodulin coimmunoprecipitates with endogenous Nav1.8 channels from native DRG, suggesting that the two proteins can interact in vivo. Treatment of native DRG neurons with a calmodulin-binding peptide (CBP) reduced the current density of Nav1.8 by nearly 65%, without changing voltage dependency of activation or steady-state inactivation. To investigate the functional role of CaM binding to the IQ motif in the Nav1.8 C-terminus, the IQ dipeptide was substituted by DE; we show that this impairs the binding of CaM to the IQ motif. Mutant Nav1.8IQ/DE channels produce currents with roughly 50% amplitude, but with unchanged voltage dependency of activation and inactivation when expressed in DRG neurons from Nav1.8-null mice. We also show that blocking the interaction of CaM and Nav1.8 using CBP or the IQ/DE substitution causes a buildup of inactivated channels and, in the case of the IQ/DE mutation, stimulation even at a low frequency of 0.1 Hz significantly enhances the frequency-dependent inhibition of the Nav1.8 current. This study presents, for the first time, evidence that calmodulin associates with a sodium channel, Nav1.8, in native neurons, and demonstrates a regulation of Nav1.8 currents that can significantly affect electrogenesis of DRG neurons in which Nav1.8 is normally expressed.
钠通道Nav1.8产生一种缓慢失活、对河豚毒素耐受的电流，其特征是从失活状态恢复时有快速和慢速成分，并且在背根神经节（DRG）神经元动作电位去极化阶段的电流中占很大比例。Nav1.8的C末端带有一个保守的钙调蛋白结合异亮氨酸 - 谷氨酰胺（IQ）基序。我们在此表明，钙调蛋白与天然DRG中的内源性Nav1.8通道共免疫沉淀，这表明这两种蛋白质在体内可以相互作用。用钙调蛋白结合肽（CBP）处理天然DRG神经元可使Nav1.8的电流密度降低近65%，而不改变激活或稳态失活的电压依赖性。为了研究钙调蛋白与Nav1.8 C末端IQ基序结合的功能作用，将IQ二肽替换为DE；我们表明这会损害钙调蛋白与IQ基序的结合。当在来自Nav1.8基因敲除小鼠的DRG神经元中表达时，突变型Nav1.8IQ/DE通道产生的电流幅度约为50%，但激活和失活的电压依赖性不变。我们还表明，使用CBP或IQ/DE替换来阻断钙调蛋白与Nav1.8的相互作用会导致失活通道的积累，并且在IQ/DE突变的情况下，即使以0.1 Hz的低频刺激也会显著增强Nav1.8电流的频率依赖性抑制。这项研究首次提供了证据，证明钙调蛋白在天然神经元中与钠通道Nav1.8相关联，并证明了对Nav1.8电流的调节，这种调节可显著影响正常表达Nav1.8的DRG神经元的电发生。
