Vasa recta voltage-gated Na+ channel Nav1.3 is regulated by calmodulin.
Vasa recta voltage-gated Na+ channel Nav1.3 is regulated by calmodulin.
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DOI：
10.1152/ajprenal.00070.2006
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发表时间：
2007
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影响因子：
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通讯作者：
W. Lee‐Kwon;J. Goo;Zhong Zhang;E. Silldorff;T. Pallone
中科院分区：
文献类型：
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作者：
W. Lee‐Kwon;J. Goo;Zhong Zhang;E. Silldorff;T. Pallone
大鼠下行直血管（DVR）表达河豚毒素（TTX）敏感的电压操纵Na（+）（Na（V））电导。我们检测了DVR中Na（V）亚型的表达，并测试了钙调素（CaM）对Na（V）电流的调节。在分离的透化DVR中使用靶向TTX敏感同种型的简并引物进行RT-PCR，扩增了其序列仅识别Na（V）1.3的产物。外髓匀浆的免疫印迹证实Na（V）1.3表达，荧光免疫化学显示Na（V）1.3在分离的血管中表达。外髓连续切片的免疫化学证实Na（V）1.3仅限于α-平滑肌肌动蛋白阳性血管束。Na（V）1.3具有COOH末端的CaM结合基序。使用下拉分析和免疫沉淀实验，我们验证了钙调素结合全长Na（V）1.3或GST-Na（V）1.3 COOH末端融合蛋白。在膜片钳实验中，Na（V）电流被钙调蛋白抑制肽（CIP; 100 nM）或CaM抑制剂N-（6-氨基己基）-5-氯-1-萘磺酰胺盐酸盐（W7）抑制。CIP和W7均未改变周细胞Na（V）电流的电压依赖性;然而，将电极游离Ca（2+）从20 nM升高至约2，000 nM会产生激活的去极化偏移。CaM与GST-Na（V）1.3C的体外结合不受Ca（2+）浓度的影响。我们的结论是Na（V）1.3由DVR表达，与CaM结合，并受CaM和Ca（2+）的调节。钙调素结合的抑制抑制周细胞钠（V）电流。
Rat descending vasa recta (DVR) express a tetrodotoxin (TTX)-sensitive voltage-operated Na(+) (Na(V)) conductance. We examined expression of Na(V) isoforms in DVR and tested for regulation of Na(V) currents by calmodulin (CaM). RT-PCR in isolated permeabilized DVR using degenerate primers targeted to TTX-sensitive isoforms amplified a product whose sequence identified only Na(V)1.3. Immunoblot of outer medullary homogenate verified Na(V)1.3 expression, and fluorescent immunochemistry showed Na(V)1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that Na(V)1.3 is confined to alpha-smooth muscle actin-positive vascular bundles. Na(V)1.3 possesses a COOH-terminal CaM binding motifs. Using pull-down assays and immunoprecipitation experiments, we verified that CaM binds to either full-length Na(V)1.3 or a GST-Na(V)1.3 COOH-terminal fusion protein. In patch-clamp experiments, Na(V) currents were suppressed by calmodulin inhibitory peptide (CIP; 100 nM) or the CaM inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride (W7). Neither CIP nor W7 altered the voltage dependence of pericyte Na(V) currents; however, raising electrode free Ca(2+) from 20 to approximately 2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-Na(V)1.3C was not affected by Ca(2+) concentration. We conclude that Na(V)1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and Ca(2+). Inhibition of CaM binding suppresses pericyte Na(V) currents.