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 1

Identification of voltage-gated K+ channel proteins as binding partners for L-CSNO.

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Identification of voltage-gated K+ channel proteins as binding partners ...
(A) Method 1. Murine cortex membrane proteins (50 μg/ lane; 2 sets of experiments using 1 C57BL/6 WT mouse) underwent native PAGE, then were incubated (30 minutes; dark; 27°C) with L-CSNO (50 μM) with or without 100 μM each L-CSMe and L-CSφ. Rinsed gels were developed with 40 μM DAF2 (30 minutes; dark; 27°C), and fluorescing bands (arrow) analyzed by LC-MS (compared with the control lane). Note that, because this is native PAGE, proteins were not separated before electrophoresis. (B) Method 2. Cysteine (100 mM) coupled to AminoLink Plus resin (4 hours, 27°C) or resin alone was incubated with EtONO (10 minutes; dark; 27°C). Pink color demonstrates S-nitrosothiol formation on the column (37). (C) Membrane proteins as in A (different animal) were loaded on columns (B) (30 min; dark; 27°C), washed, then eluted with Laemmli buffer followed by 0.1 M glycine, pH 3.5. Eluate underwent SDS-PAGE, and bands (including that shown by the arrow, 20 kDa) were analyzed by LC-MS in comparison with the control lane. Both methods identified several Kv channel proteins (see Supplemental Tables 2 and 3). Note that, because native PAGE was used (proteins were not separated before electrophoresis) in A, and a broad region of discordance was excised in C, multiple molecular weight proteins were evident by LC-MS. (D) Proteins as in A from WT and Kv1.1–/– mice (2 sets of experiments using WT and C57BL/6 background mice) were loaded on and eluted from columns as in B and C, and immunoblotted for Kv1.1 and Kvβ2.