Vasa recta voltage-gated Na+channel Nav1.3 is regulated by calmodulin
| Autor: | Jae Hwan Goo, Whaseon Lee-Kwon, Erik P. Silldorff, Thomas L. Pallone, Zhong Zhang |
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| Rok vydání: | 2007 |
| Předmět: |
Male
Gene isoform medicine.medical_specialty Patch-Clamp Techniques Calmodulin Physiology Blotting Western Fluorescent Antibody Technique Nerve Tissue Proteins In Vitro Techniques Sodium Channels Renal Circulation Rats Sprague-Dawley chemistry.chemical_compound Internal medicine NAV1.3 Voltage-Gated Sodium Channel medicine Animals Immunoprecipitation Patch clamp Enzyme Inhibitors Egtazic Acid Chelating Agents Kidney Medulla Sulfonamides Voltage-gated ion channel biology Reverse Transcriptase Polymerase Chain Reaction Vasa recta Glutathione Rats Cell biology Electrophysiology Endocrinology medicine.anatomical_structure chemistry NAV1 Tetrodotoxin biology.protein Blood Vessels Ion Channel Gating Plasmids |
| Zdroj: | American Journal of Physiology-Renal Physiology. 292:F404-F414 |
| ISSN: | 1522-1466 1931-857X |
| DOI: | 10.1152/ajprenal.00070.2006 |
| Popis: | Rat descending vasa recta (DVR) express a tetrodotoxin (TTX)-sensitive voltage-operated Na+(NaV) conductance. We examined expression of NaVisoforms in DVR and tested for regulation of NaVcurrents by calmodulin (CaM). RT-PCR in isolated permeabilized DVR using degenerate primers targeted to TTX-sensitive isoforms amplified a product whose sequence identified only NaV1.3. Immunoblot of outer medullary homogenate verified NaV1.3 expression, and fluorescent immunochemistry showed NaV1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that NaV1.3 is confined to α-smooth muscle actin-positive vascular bundles. NaV1.3 possesses a COOH-terminal CaM binding motifs. Using pull-down assays and immunoprecipitation experiments, we verified that CaM binds to either full-length NaV1.3 or a GST-NaV1.3 COOH-terminal fusion protein. In patch-clamp experiments, NaVcurrents 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 NaVcurrents; however, raising electrode free Ca2+from 20 to ∼2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-NaV1.3C was not affected by Ca2+concentration. We conclude that NaV1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and Ca2+. Inhibition of CaM binding suppresses pericyte NaVcurrents. |
| Databáze: | OpenAIRE |
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