Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling
Benjamin Gaston, Laura Smith, Jürgen Bosch, James Seckler, Diana Kunze, Janna Kiselar, Nadzeya Marozkina, Craig A. Hodges, Patrick Wintrobe, Kellen McGee, Tatiana S. Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis
Benjamin Gaston, Laura Smith, Jürgen Bosch, James Seckler, Diana Kunze, Janna Kiselar, Nadzeya Marozkina, Craig A. Hodges, Patrick Wintrobe, Kellen McGee, Tatiana S. Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis
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Research Article Cell biology

Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling

Abstract

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase–independent (sGC-independent) effects are stereoselective — that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) — and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays — followed by unbiased proteomic analysis — to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO–Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvβ2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.

Authors

Benjamin Gaston, Laura Smith, Jürgen Bosch, James Seckler, Diana Kunze, Janna Kiselar, Nadzeya Marozkina, Craig A. Hodges, Patrick Wintrobe, Kellen McGee, Tatiana S. Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis

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Figure 6

L-CSNO inhibition of voltage-gated K+ current.

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L-CSNO inhibition of voltage-gated K+ current. (A) Top view: Schematic s...
(A) Top view: Schematic showing L-CSNO’s effect of altering the structure of the Kv multimer. The HDX data (Figure 3 and Supplemental Figure 3) show that the tertiary structure of Kvβ2 is altered by L-CSNO binding to make the protein more concave, particularly at the base near the site of its interaction with Kvα proteins. Kv1.1 T1 is also affected (Figure 3 and Supplemental Figure 3), but patch clamp data suggest that the interaction with Kvβ2 is essential for a functional effect (Figure 2). L-CSNO is formed from GSNO by γ-glutamyl transpeptidase and downstream dipeptidases (1), signaling increased VE (1). GSNO is formed, in turn, by NOS activation, oxyhemoglobin desaturation, ceruloplasmin and other metalloproteins capable of transferring NO+ equivalents to thiolate anions. Uniquely, NO is not transferred to the Kv proteins. D-CSNO does not share the activity of L-CSNO (Figures 2–5), though both isomers form NO at the same rate (20). (B) Lateral view of the Kvα/Kvβ complex in the plasma membrane, as in Figure 2A: side view of K+ exit from the cell. (C) Based on HDX, approximate sites of L-SNO binding in the 4 Kvβ2 subunits. (D) In silico estimates of L-CSNO interaction with Kvβ2 amino acids using ChemDraw and Schrödinger Maestro software. Reproduced with permission from V. Ferrante.