A persulfidation-based mechanism controls aquaporin-8 conductance

Abstract

Upon engagement of tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases release H₂O₂ in the extracellular space. We reported previously that aquaporin-8 (AQP8) transports H₂O₂ across the plasma membrane and is reversibly gated during cell stress, modulating signal strength and duration. We show that AQP8 gating is mediated by persulfidation of cysteine 53 (C53). Treatment with H₂S is sufficient to block H₂O₂ entry in unstressed cells. Silencing cystathionine β-synthase (CBS) prevents closure, suggesting that this enzyme is the main source of H₂S. Molecular modeling indicates that C53 persulfidation displaces a nearby histidine located in the narrowest part of the channel. We propose that H₂O₂ molecules transported through AQP8 sulfenylate C53, making it susceptible to H₂S produced by CBS. This mechanism tunes H₂O₂ transport and may control signaling and limit oxidative stress.

Fig. 1. H₂S blocks AQP8-dependent H₂O₂ transport.

(A to C) The kinetics of HyPerCyto activation in HeLa cells upon addition of 50 μM H₂O₂ and with or without a pretreatment with H₂S (blue and red traces, respectively). Colored arrows indicate the time at which indicated compounds were added to cells (5 mM DTT, black; 5000 U/ml of catalase, green; and 10 mM MESNA, brown). (D) Time course of HyPer activation in HeLa cells expressing either HaloAQP8 WT (left graph) or the C53S mutant (right graph) upon addition of 50 μM H₂O₂ and treated with or without H₂S. Only cells expressing the transgene were analyzed [see (E)]. In all cases (A to D), data are shown as mean fold changes of the 488/405-nm ratio measured by confocal laser scanning, plotted against time. Mean of ≥3 experiments are shown ± SEM. Ctrl, control. (E) Frames extracted from time course analyses of H₂O₂ import on cells expressing WT or C53S HaloAQP8 treated with H₂S. Cells expressing the transgene are stained by fluorescent Halo ligands (upper frames). The 488/405-nm ratio intensity, represented in pseudocolor, reflects the activation of HyPerCyto upon addition of 50 μM H₂O₂ (lower frames). Note that the sensor is activated only in C53S-expressing cells. Scale bars, 50 μm.

Fig. 2. CBS produces H₂S for AQP8 gating in heat-stressed cells.

(A) H₂O₂ transport was analyzed as in Fig. 1 in HeLa cells expressing low (CBSi) or normal (CBS WT) levels of CBS. H₂O₂ entry into CBSi HeLa cells was no longer inhibited by heat stress (light blue trace), but treatment with H₂S restored inhibition (green trace). Mean of ≥3 experiments ± SEM. (B) Lysates from HeLa transfectants expressing a Flag-tagged HaloAQP8myc (AQP8), calnexin-Flag (Cn), or Halo (Ha) were immunoprecipitated with anti-Flag beads and immunoblotted with the indicated antibodies. WCL, whole cell lysate; IP, immunoprecipitation. (C) Aliquots from lysates of HeLa cells kept at 42°C for increasing times were blotted with the indicated antibodies. Note the appearance of a cleaved CBS form after heat shock.

Fig. 3. Radioactive labeling of AQP8 persulfidation.

(A) The scheme summarizes the protocol used to detect C53 persulfidation. Proteins can be radiolabeled by incorporating ³⁵S methionine and cysteine as building blocks during protein synthesis (right branch) or by reacting with H₂³⁵S produced by the transsulfuration pathway (left branch). In the presence of protein synthesis inhibitors (0.5 mM CHX), the latter pathway should be favored, especially in conditions of stress that activate CBS. Unlike radioactive amino acids incorporated into proteins, −³⁵SH persulfide moieties are removed by a 200 mM DTT treatment, allowing discrimination from radioactive amino acids that are part of the polypeptide backbone. (B) Cells transfected with either WT or C53S HaloAQP8mycFlag were kept for 3 hours at 37° or 42°C (− or + heat, as indicated) in the presence of ³⁵S amino acids and CHX (0.5 mM). AQP8 was immunoprecipitated using anti-Flag beads, resolved under nonreducing or reducing conditions (left and right, respectively), and developed to detect the ³⁵S or Halo ligand signals to normalize protein synthesis and loading. The ratio between the ³⁵S and Halo ligand signals was calculated for each sample and values normalized to the values obtained with C53S (see Material and Methods for more details), which should not be modified by H₂³⁵S (bottom). A significant difference between WT and C53S emerges only when samples are resolved under nonreducing conditions. Mean of ≥3 experiments ± SEM. *P < 0.05. (C) Scheme of the stepwise chemical reactions leading to persulfidation. (D) Quantification of the H₂O₂ uptake performed 2.5 min after addition of 50 μM exogenous H₂O₂ to HyPerCyto-HeLa cells, treated with or without 10 μM DPI for 2.5 hours before and during incubation (30 min) with H₂S. Data were normalized relative to untreated samples. Mean of ≥3 experiments ± SEM.

Fig. 4. Molecular modeling of AQP8.

(A) Sequence alignment of hAQP8 and two other H₂O₂-transporting AQPs (AQP3 and AQP9) with AQP5 (template for homology modeling). Completely conserved residues are highlighted with a red background, highly conserved amino acids colored in red, and highly homologous residues were boxed. The two highly conserved NPA motifs are colored in magenta, the Cys53 is highlighted in yellow, and residues constituting the selectivity filter region (ar/R) are indicated with cyan triangles. (B and C) Pore profile of AQP8 calculated with HOLE represented as a function of the channel relative position (B) or as multicolored surface (C), illustrating the internal surface of the pore. Green color denotes a pore radius sufficiently large for the passage of a single H₂O₂ molecule. Blue denotes a radius that allows the passage of two H₂O₂ molecules. In (B) and (C), the (ar/R) constriction region and the NPA motif of AQP8 are identified by a cyan and magenta bar, respectively, that goes across both panels. In (C), Cys53 is shown with yellow sticks and the residues forming part of the ar/R or the NPA filters with cyan and magenta sticks.

Fig. 5. Role of the amino acids surrounding the constriction site in AQP8 gating.

(A) The (ar/R) constriction site of AQP8 before (left) or after (right) persulfidation are shown. Protein and residues determining the selectivity filter are represented in cartoon and sticks, respectively. C53, R213, and H72 are highlighted using dots representation. (B) Quantification of the H₂O₂ uptake performed 2.5 min after addition of 50 μM exogenous H₂O₂ to HyPerCyto-HeLa cells transfected with different mutants and subjected to heat shock. Data were normalized to the uptake of the respective unstressed samples. Mean of ≥3 experiments ± SEM. n.s., not significant.

Fig. 6. Two-step inhibition model.

(A) The scheme summarizes the sequential chemical reactions that lead to AQP8 gating and the ways to inhibit some key steps. (B) The left panel depicts AQP8 at steady state, in which C53 is maintained as a thiolate by the positive charge of R213. When H₂O₂ flows through the channel (middle), C53 is sulfenylated, becoming susceptible for further modification by H₂S. When excess H₂O₂ accumulates, like for instance in stressed cells, CBS is activated to produce H₂S inducing, persulfidation of oxidized C53 (right). Elongation of the C53 side chain alters the conformation of the (ar/R) constriction site, presumably leading the aromatic ring of H72 to gate AQP8.