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.
Gene Ontology Annotations
