Hydrogen sulfide regulates hippocampal neuron excitability via S-sulfhydration of Kv2.1

Hydrogen sulfide (H2S) is gaining interest as a mammalian signalling molecule with wide ranging effects. S-sulfhydration is one mechanism that is emerging as a key post translational modification through which H2S acts. Ion channels and neuronal receptors are key target proteins for S-sulfhydration and this can influence a range of neuronal functions. Voltage-gated K+ channels, including Kv2.1, are fundamental components of neuronal excitability. Here, we show that both recombinant and native rat Kv2.1 channels are inhibited by the H2S donors, NaHS and GYY4137. Biochemical investigations revealed that NaHS treatment leads to S-sulfhydration of the full length wild type Kv2.1 protein which was absent (as was functional regulation by H2S) in the C73A mutant form of the channel. Functional experiments utilising primary rat hippocampal neurons indicated that NaHS augments action potential firing and thereby increases neuronal excitability. These studies highlight an important role for H2S in shaping cellular excitability through S-sulfhydration of Kv2.1 at C73 within the central nervous system.

Current awareness of the biological roles of endogenous hydrogen sulfide (H 2 S) is expanding rapidly, as our understanding of its production and cellular targets continue to develop [1][2][3] . Enzymatic production of H 2 S from cysteine is regulated by cystathionine γ lyase (CSE) and cystathionine β synthetase (CBS). With reference to the central nervous system (CNS) 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase has also been demonstrated to generate H 2 S in the brain 4,5 , with additional H 2 S protein-bound sulphur "stores" 6 . H 2 S levels in the brain have been reported in the µM range 7,8 , however real time measurements are challenging given the physiochemical nature of the gasotransmitter [9][10][11] . While physiological levels are known to mediate diverse signalling cascades, there is the potential to develop cellular toxicity however a consensus on the molecular basis of H 2 S mediated neurotoxicity is lacking 12,13 . Furthermore, some effects of H 2 S have been demonstrated to occur through polysulfides (H 2 S n ) which are important physiological signalling species in themselves 14,15 .
In the CNS, the gasotransmitter, H 2 S promotes induction of long-term potentiation 7 , modifies astrocytic Ca 2+ signalling 16 and protects neurons against oxidative stress 17 . Given the diversity of effects, it is perhaps surprising that one mechanism of action is emerging as vital in regulating these processes; the modification of protein thiol groups termed S-sulfhydration, in which the thiol -SH groups in cysteine residues are converted to persulfide -SSH groups 1,18 , leading to modulation of protein function and/or protection against oxidation. Such a posttranslational modification is analogous to the widespread S-nitrosylation of diverse proteins by nitric oxide 19 . Studies have suggested that up to 50% of cellular proteins may be basally S-sulfhydrated 1,18 , implying that this post-translational modification is of widespread physiological importance.
Results are presented as means ± S.E.M., and statistical analysis performed using unpaired Student's t-tests where P < 0.05 was considered statistically significant.
Modified biotin switch assay. The assay was carried out as described previously 18  www.nature.com/scientificreports/ UK). Biotin-N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamide (HPDP) was added to the buffer exchanged protein samples a to a final concentration of 4 mM and incubated in the dark for 90min at room temperature with constant shaking (1400 rpm), biotinylated proteins were then buffer exchanged using spin columns to remove biotin-HPDP in the neutralization buffer (HEPES 20 mM; NaCl 100 mM; EDTA 1 mM Triton X-100 0.5%, pH 7.7) and precipitated by streptavidin-agarose beads, for 16 h at 4 °C. The biotinylated proteins were eluted by SDS-PAGE sample buffer and subjected to Western blot analysis. Anti-Kv2.1 antibodies K89/34 (NeuroMab, Davis, CA) were used to detect Kv2.1 WT and C73A. β-Actin was used as loading control (Sigma-Aldrich, UK). ImageJ software (NIH, Bethesda, USA) was used to analyse band intensities for Kv2.1 before and after the assay.
Hydrogen sulfide generation. Hydrogen sulfide (H 2 S) was produced using the donor molecule sodium hydrosulfide (NaHS, Sigma-Aldrich, UK) and made up as a stock solution in Hank's Balanced Salt Solution. This stock was then diluted in the perfusate to give the working concentration as stated in the text. To avoid degradation and loss of H 2 S the stocks were routinely made up immediately prior to experimentation. In addition, to confirm a role for H 2 S, the H 2 S-slow releasing molecule P-(4-methoxyphenyl)-p-4-morpholinylphosphinodithioic acid morpholine salt (GYY4137; Sigma-Aldrich, UK) was used. GYY4137 (1 M stock) was dissolved in DMSO and stored at − 80 °C, on the day of experiments the stock was diluted to 100 mM in perfusate and used on the same day, the final concentration of DMSO in the bath did not exceed 0.001%, when higher GYY4137 concentrations were used a 20 mM stock was made fresh in PBS and used on the same day for these experiments.

Results
To examine whether Kv2.1 could be regulated by H 2 S, we firstly recorded whole-cell currents from HEK293 cells stably expressing recombinant Kv2.1, as previously described 28 . Bath application of the H 2 S donor, NaHS (20 μM-1 mM), inhibited currents flowing through Kv2.1 channels in a concentration-dependent manner (Fig. 1A,B,C). Currents were inhibited at all activating test potentials (Fig. 1B) and there was no significant effect of NaHS on whole-cell conductance (Fig. 1C), suggesting that inhibition of current amplitudes was not attributable to altered gating. Inhibition of Kv2.1 currents was largely irreversible upon washout (e.g. Fig. 1E; 23.5 ± 4.1% recovery after 10 min, n = 12). However, current amplitudes could be recovered to near pre-NaHS treatment levels by exposure to the reducing agent dithiothreitol (DTT, 1 mM; Fig. 1F, 83.4 ± 3.7% recovery, n = 13). Similar results were obtained using another H 2 S donor molecule GYY4137 (Fig. 2). Kv2.1 currents at test potentials greater than + 40 mV by 30 µM GYY4137 ( Fig. 2A,B), this concentration was determined following a dose-response relationship for the effects of the H 2 S donor GYY4137 (Fig. 2D). Normalized current amplitudes was inhibited by 40.1 ± 8.2%, n = 15, measured at + 50 mV and this inhibition was irreversible upon washout (Fig. 2C).
Reversal of H 2 S inhibition of Kv2.1 by DTT suggested a possible involvement of redox regulation of the channel protein. Sesti and colleagues 29 have demonstrated that cysteine 73 is crucial to the response of this channel to oxidation, and so we generated a C73A mutant to explore the role of this residue in H 2 S regulation. As exemplified in Fig. 3A, and quantified in Fig. 3B, NaHS was without effect on the C73A mutant, whilst wild-type channels, studied in parallel, were significantly inhibited. Kv2.1 protein has an array of modifiable cysteine residues, some of which are not sensitive to oxidative modification 29 . To determine a role for other cystine residues in the observed NaHS effect, we generated several other mutant cell lines. C29A and C831A still provided functional channels however both cell lines were sensitive to inhibition following application of 250 μM NaHS (C831A by 20 ± 2% (n = 6); C29A by 26.4 ± 4% (n = 5), (Fig. 3C,D).
Many of the diverse effects of H 2 S in different systems arise due to specific, direct modulation of target proteins by S-sulfhydration (i.e. the conversion of -SH groups in cysteine residues into -SSH groups). S-sulfhydration is detectable via by the modified biotin switch assay 18 , and this approach was taken with the recombinant Kv2.1 protein. As illustrated in Fig. 4A, NaHS did indeed S-sulfhydrate recombinant Kv2.1, evident from 30 mins, suggesting that the mechanism of channel inhibition was due to direct protein modification (full blots can be found as Supplementary Figures S1, 2 online). This was supported by the observation that the C73A mutant form of the channel appeared resistant to this form of post-translational modification ( Fig. 4A; full blots available as Supplementary Figure S3 online). To provide a level of quantification 50.8 ± 7.5%, (n = 3) of WT Kv2.1 channel protein (Fig. 4B) can be identified as S-sulfhydrated after 2 h treatment with NaHS (250 µM) following modified biotin switch assay (time dependence further explored in Supplementary Figure S4 online). The time discrepancies observed between functional and biochemical experimentation are likely to reflect technical limitations of single cell versus population studies. The combination of these methodologies has been extensively used when examining post-translational modifications of plasma membrane proteins.
Kv2.1 is widely expressed in the CNS, and its ability to control the firing pattern of hippocampal neurons in particular has been described in detail (reviewed in Ref. 25 ). As with HEK293 cells, we employed whole-cell patch-clamp recordings to demonstrate that NaHS caused significant inhibition of K + currents in hippocampal rat neurons (Fig. 5A,B), at all activating test potentials studied. As was the case for recombinant Kv2.1, NaHS-mediated inhibition of K + current amplitudes in hippocampal neurons could be recovered by DTT (1 mM; Fig. 5C).
Numerous different voltage-gated K + currents contribute to the whole-cell K + current in these cells. We and others have previously demonstrated that the contribution to the whole-cell current by Kv2.1 can be determined by intracellular dialysis (via the patch pipette) of an anti-Kv2.1 antibody 28,30 . In agreement with our earlier studies, intracellular dialysis with this antibody reduced current amplitudes by around 50% (Fig. 5D). Subsequent exposures to NaHS (100 µM) did not significantly reduce currents further (Fig. 5D), indicating that NaHS was selective in its ability to inhibit Kv2.1 despite the presence of other voltage-gated K + channels in hippocampal neurons.  , except that, following washout of NaHS, the cell was exposed to dithiothreitol (DTT, 1 mM) for the period indicated by the second horizontal bar. Inset shows example currents under the conditions indicated (DTT applied after washout of NaHS). Scale bars: 2 nA (vertical) and 10 ms (horizontal). www.nature.com/scientificreports/ cells (9.1 ± 1.8 mV, n = 6, P < 0.01), indicative of reported H 2 S effects on TRP channels in dorsal root ganglion neurons 31 . This effect was compensated by current injection, in order to examine the effects of depolarizing current injections in cells with a standardised membrane potential of − 70 mV (Fig. 5A,B). Square wave current injections evoked action potentials which increased in frequency with increasing current amplitude (Fig. 5A,B).
In the presence of NaHS (200 µM), action potential frequency was augmented, particularly following larger current injections, as anticipated when Kv2.1 channels participate in neuronal excitability.

Discussion
Our study identifies H 2 S as another important modulator of the voltage-gated K + channel Kv2.1. This channel is highly expressed in the cortex and hippocampus where it is a major determinant of intrinsic neuronal excitability through a diverse complement of signalling cascades. High levels of constitutive phosphorylation determine the voltage-dependence of gating and location of Kv2.1, and both factors can influence neuronal excitability in a dynamic manner 25,32,33 . Activity-dependent, calcineurin-mediated dephosphorylation of Kv2.1 can suppress excitability 33 , and rapid rephosphorylation (e.g. via CDK5 34 ) can restore excitability. However, this is dependent on the location of the phosphorylation site within the channel protein: for example, AMP-activated protein kinase-mediated phosphorylation at distinct residues (S440 and S537) can suppress excitability and exert hyperpolarizing shifts in the voltage-dependent activation, similar to calcineurin-mediated dephosphorylation of other residues 27 . In addition to alteration of gating via phosphorylation, Kv2.1 channel inhibition www.nature.com/scientificreports/ (e.g. following antisense knockdown) also increases high frequency excitability 35,36 . The present study indicates that this phenomenon also occurs because of channel inhibition by H 2 S (Fig. 3). Thus, using larger depolarizing current injections, action potential frequency was augmented by H 2 S, suggesting that this gasotransmitter may influence excitability physiologically through modulation of Kv2.1 selectively. It remains to be determined if polysulfides mediated this inhibition as has been reported for Kv1.4 and Kv3.4 23 . This is likely to be mediated via S-sulfhydration of the channel given our biochemical evidence. There are time discrepancies between the acute exposure in the functional recordings and the biochemical observations, but these are comparable to other reports of ion channel post-translational modification (see Ref. 18 ). This likely reflects the number of S-sulfhydrated channels required to produce a functional effect versus the amount of S-sulfhydrated protein detected via our biochemical experimentation. Such complex regulation of a single channel target suggests it is of fundamental importance to the excitability of neurons in which it is expressed, but the fact that it is also a target for modulation by gasotransmitters supports the belief that it has even greater physiological significance. While evidence exists that other Kv channels are modulated by H 2 S, such as Kv7 24,37,38 , the neuronal localisation of these channels will ultimately determine their influence on action potential dynamics. For example, Kv7 activation via NaHS has been reported to suppress neuropathic pain 24 , indicative of a decrease in network excitability 39 . These studies have implicated the H 2 S in the mechanisms of neuropathic pain mediated by the Kv7 family. This furthers the diversity of ion channels targeted www.nature.com/scientificreports/ by H 2 S and also raises questions over selectivity 20 . With respect to the hippocampus, Kv7 channels has been localised to the axonal initial segment 40 in contrast Kv2.1 channels display a predominant somatodendritic loci 41 . Therefore, discrete localisation of Kv channels 42, 43 may underpin the respective action of H 2 S on neuronal output.
Kv2.1 has also been proposed to play a vital role in oxidative stress-induced apoptosis of central neurons: oxidants can initiate apoptosis in a Zn 2+ -dependent manner which leads to co-ordinated, Src kinase-mediated phosphorylation of the channel protein at an N-terminal tyrosine (Y124) together with p38 MAPK-mediated phosphorylation at a C-terminal site (S800). This in turn leads to rapid channel insertion into the plasma membrane; the resultant loss of cytosolic K + triggers caspase activation and apoptosis. The present study suggests H 2 S may provide protection against oxidant-induced apoptosis via inhibition of Kv2.1. Indeed, we have previously shown that Kv2.1 is inhibited by carbon monoxide (CO), which is derived from heme oxygenases (HO-1 and HO-2). The inducible heme oxygenase, HO-1, is upregulated by numerous stress factors and its ability to provide protection against apoptosis in hippocampal neurons arises, at least in part, from the ability of its product, CO,  www.nature.com/scientificreports/ to inhibit Kv2.1 28 . Interestingly, a similar Kv2.1-mediated protective mechanism may be active in some forms of cancer (in which resistance to apoptosis is a hallmark feature of the disease 44 ) where HO-1 expression is constitutively high 45 .
Interestingly, research indicates that oxidative stress can modify Kv2.1 channels via a novel means, causing their oligomerization through formation of disulfide bridges with adjacent channel proteins. This in turn leads to channel clustering and promotion of apoptosis 29 , and it was proposed that this is an important aspect of both normal, age-related loss of neuronal function which is exacerbated in Alzheimer's disease 29 . Animal studies also highlight modulation of the Kv2.1 protein function, to increase hippocampal neuronal excitability, in the 3xTg-AD model of Alzheimer's disease 46 . In this regard, channel inhibition by H 2 S may be of particular importance: the modified biotin switch assay revealed that H 2 S caused S-sulfhydration of the channel protein (Fig. 2C), and this has been proposed to provide protection of vulnerable cysteine residues against oxidation 1 . This could have implications for cognitive decline and supports the use of H 2 S donors to modulate Alzheimer's pathology [47][48][49] however further consideration of the temporal profile of pathology alongside H 2 S administration is required.
In summary, we have shown that H 2 S inhibits both recombinant and native Kv2.1 and propose that this occurs via S-sulfhydration of the channel protein. This occurred in a concentration dependent manner; however, we cannot rule out the interactions of other proposed S-sulfhydrated molecular targets on our neuronal observations. However, consistent with the known physiological role of Kv2.1, we show that H 2 S-mediated inhibition contributes to augmentation of higher rates of evoked action potential frequencies with the Kv2.1 protein acting as a molecular rheostat 50 . This has implications for pathological conditions where (i) H 2 S production is altered (e.g. dementia 51 ) or (ii) where the Kv2.1 protein has been oxidised at C73 (e.g. ageing 29 ). Future studies will determine whether S-sulfhydration of Kv2.1 will provide protection against apoptosis arising from oxidative stress, for example, because of aging or neurodegenerative diseases.