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
View: Text | PDF
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

×

Figure 2

SyncroPatch analysis of the Shaker channel constructs.

Options: View larger image (or click on image) Download as PowerPoint
SyncroPatch analysis of the Shaker channel constructs. (A) Kv1.2/Kvβ2 cr...
(A) Kv1.2/Kvβ2 crystal structure (PDB 2A79). The homotetramer of Kv1.2 is in the brown ribbon; Kvβ2 is the green surface; the plasma membrane (PM) is gray. (B) Schematic drawing of the individual cell lines after transfection with Kv1.1, Kv1.2, and Kvβ2. Not all cells will express all proteins; this schematic shows all permutations, with the corresponding experimental patch clamp results. Icons surrounded by red boxes show no conductance, while icons with a light green background show specific K+ channel activity. Gray boxes indicate possible conformations that cannot be distinguished based on conductance. (C) Conductance characteristics of the different cells lines upon voltage gate clamping from –120 mV to +80 mV (n = 204 single-cell patch clamp studies, with 20 untransformed, 20 Kvβ2 alone, 26 Kv1.1 alone, 28 Kv1.2 alone, 28 Kv1.1/Kvβ2, 25 Kv1.2/Kvβ2, 27 Kv1.1/Kv1.2, and 61 triple-expressing). Current at each voltage above 20 mV is greater for the triple-overexpressing than for the other cells.) (D) Conductance of each cell line at +80 mV resting potential. Note that only Kv1.1/Kv1.2 and Kv1.1/Kv1.2/Kvβ2 show currents >500 pA. n is the same as described in C. (E) Examples of individual traces, showing 11 sweeps corresponding to the ramping protocol for different cells. Black is +80 mV. All traces are on the same scale. (F) Effect of L-SNOC on K+ current block in either the double or triple cell line. n = 8 cells in each condition; P = 0.002 by Mann-Whitney rank-sum test t test. (G) IC50 determination of L-CSNO on triple cell line with currents >500 pA represented as % block (error bars represent a confidence level of 95% [CL 95]) compared with TEA full block. There is no block with either vehicle (green) or D-CSNO (red). n = 8 in the L-CSNO group and n = 8 in the D-CSNO group. At concentrations greater than 500 nM (n = 236 points analyzed), inhibition by L-CSNO is greater than inhibition by D-CSNO (P = 0.0001 vs. D-CSNO and P = 0.0048 vs. vehicle, by Kruskal-Wallis test). Data are presented as median ± 95% CI.