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Vasa recta voltage-gated Na⁺ channel Na_v1.3 is regulated by calmodulin

¹Division of Nephrology, Department of Medicine, and ²Department of Physiology, University of Maryland School of Medicine, Baltimore; and ³Department of Biology, Towson University, Towson, Maryland

Submitted 24 February 2006 ; accepted in final form 10 August 2006

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_V1.3. Immunoblot of outer medullary homogenate verified Na_V1.3 expression, and fluorescent immunochemistry showed Na_V1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that Na_V1.3 is confined to -smooth muscle actin-positive vascular bundles. Na_V1.3 possesses a COOH-terminal CaM binding motifs. Using pull-down assays and immunoprecipitation experiments, we verified that CaM binds to either full-length Na_V1.3 or a GST-Na_V1.3 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 Ca²⁺ from 20 to 2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-Na_V1.3C was not affected by Ca²⁺ concentration. We conclude that Na_V1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and Ca²⁺. Inhibition of CaM binding suppresses pericyte Na_V currents.

kidney; medulla; microcirculation; patch clamp; channel

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