Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)

Abstract

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP₂ interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H₂S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H₂S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H₂S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H₂S regulation depended on the strength of the channel-PIP₂ interactions. H₂S regulation was attenuated when channel-PIP₂ interactions were strengthened and was increased when channel-PIP₂ interactions were weakened by depleting PIP₂ levels. These H₂S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H₂S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H₂S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP₂ interactions and channel gating.

Keywords: GIRK channels; KATP channels; Kir3; Kir3 or GIRK channels; gasotransmitters; hydrogen sulfide; inwardly rectifying K+ (Kir) channels; ischemia; phosphatidylinositol 4,5-bisphosphate (PIP2); phosphoinositide; potassium channel; stroke.

Figure 1.

Exogenous H₂S inhibits Kir3.2 channel activity. A, representative currents elicited by voltage ramps from oocytes treated with 2 mm NaHS for 100 min versus untreated controls (0-min treatment) show 35.2% (12.5 to 8.1 μA) inhibition of Kir3.2* homomeric channel activity at −80 mV. B, NaHS incubation of Kir3.2*-expressing oocytes yielded maximal inhibition by 100 min from −11.68 ± 2.80 to −6.07 ± 1.40 μA (**, p < 0.01) at a test potential of −80 mV. C, NaHS-treated currents are normalized to vehicle (untreated controls). Incubation of Kir3.2* channels with 2 mm NaHS elicits time-dependent inhibition. Treatment with NaHS at increasing intervals leads to the following normalized current values: 1.00 ± 0.39 (n = 20) at 0 min, 0.96 ± 0.51 (n = 20) at 15 min, 0.72 ± 0.05 (n = 20) at 60 min, 0.46 ± 0.19 (n = 25) at 100 min, and 0.43 ± 0.27 (n = 17) at 400 min, and they were assessed for statistical significance using one-way repeated-measures ANOVA and the Holm-Sidak post-test (p < 0.05). Post-incubation, Kir3.2* activity is maximally inhibited by ∼45% at 100 min (and not further inhibited at 400 min). D, incubation with NaHS does not affect surface localization of Kir3.2*. Data summary (mean ± S.D.) of Kir3.2 surface localization levels was quantified by chemiluminescence as detailed under “Experimental procedures.” Data were obtained from four independent experiments and were assessed for statistical significance using an unpaired Student's t test. Negative control (Neg. ctrl) values were less than 0.5%. The total numbers of oocytes analyzed in each of the 0, 100 min, and negative control groups were 69, 62, and 69, respectively. **, p < 0.01; N.S., non-significant; m, min.

Figure 2.

Exogenous H₂S inhibits Kir3.2 channels in mammalian cells by decreasing the open probability. A, whole-cell currents recorded from CHO-K1 cells expressing Kir3.2* are decreased by 100 μm NaHS. Recording protocols are described under “Experimental procedures.” B, 300-s treatment with 100 μm NaHS reduces Kir3.2* channel currents in CHO-K1 cells by ∼40% (n = 8 cells) and Kir3.2-mediated currents by ∼60% (n = 10) and assessed for statistical significance using an unpaired Student's t test; **, p < 0.01. C, NaHS reduces Kir3.2* channel activity in a time-dependent manner. Currents were elicited by test pulses to −120 mV from a holding level of −80 mV every 10 s. Treatment with 100 μm decreased the current by ∼40% within 300 s (n = 8), an effect that was stable when NaHS was washed out. D, left, example images of CFP-Kir3.2 channels at the surface of CHO-K1 cells, captured by total internal reflection fluorescence microscopy. The mean surface-density of CFP-pixels was assessed every 100 s following exposure to 100 μm NaHS. Right, mean surface-density of CFP-pixels decreases in a non-significant (N.S.) manner over 600 s with or without 100 μm NaHS (unpaired Student's t test used to determine statistical significance between groups). E, single Kir3.2* channels were studied in cell-attached mode before and after a 300-s treatment with 100 μm NaHS. The effective voltage was −120 mV. F, histograms show that the distribution of single-Kir3.2* channel amplitudes is not changed from control (upper) by a 300-s treatment with 100 μm NaHS (lower). The histograms are fit with a Gaussian function (black line) to determine the mean (±σ) single channel amplitudes of 1.2 ± 0.26 pA (∼30 pS) before and 1.18 ± 0.25 pA (∼30 pS) after treatment with 100 μm NaHS, based on >80,000 events per condition. G, dwell time histograms for single Kir3.2* channels show that the distribution of open times (left) is decreased by a 300-s treatment with 100 μm NaHS, while the closed time is increased. Data were calculated from >80,000 events per condition. N.S., non-significant.

Figure 3.

H₂S effects on Kir channel activity. A, H₂S inhibits cardiac Kir3.4 (S143T) homomeric channel and Kir3.4/Kir3.1 heteromeric channel activity. Treatment with NaHS led to ∼42% inhibition of Kir3.4* channels (0.58 ± 0.20, n = 13) and ∼67% inhibition of Kir3.4/Kir3.1 channels (0.33 ± 0.14, n = 10). B, treatment with NaHS significantly inhibited Kir2.3 channels (0.64 ± 0.27, n = 17) but did not significantly alter Kir2.1 channel activity (1.01 ± 0.24, n = 13). C, currents from both groups of oocytes expressing Kir6.2/SUR2A or Kir6.2Delta36, in which the 36 C-terminal amino acids were removed from Kir6.2 to permit cell-surface expression in the absence of sulfonylurea receptor (SUR2A) subunits, increased ∼6-fold (normalized current values of 6.39 ± 1.82, n = 12, and 6.48 ± 3.98, n = 9, respectively) upon incubation with 2 mm NaHS, consistent with hyperpolarizing findings in Mustafa et al. (13), in which Kir6.1 was expressed in HEK293 cells. Unpaired Student's t test was used to assess the significant differences between NaHS-untreated (control) and NaHS-treated groups. * indicates comparison to the untreated group. Single, double, and triple symbols indicate p < 0.05, p < 0.01, and p < 0.001, respectively. N.S., non-significant.

Figure 4.

Hydrogen sulfide inhibition of Kir3.2 is altered by strengthening or weakening channel–PIP₂ interactions. A, increasing PIP₂ concentration in the oocyte (and thereby increasing channel–PIP₂ interactions) through the co-expression of PIP5K virtually abrogates H₂S inhibition of Kir3.2* channels, whereas reduction of PIP₂ levels via oocyte incubation in 25 μm Wort (a known blocker of PI4-kinases at μm concentrations) for 100 min in untreated oocytes or co-incubation (with NaHS), enhanced H₂S inhibition from ∼40% to ∼80% inhibition. B, decreasing PIP₂ though activation of co-expressed Ci-VSP, a voltage-activated PIP5 phosphatase, also reduces intracellular PIP₂, and H₂S is able to enhance channel inhibition (remaining currents from 0.43 ± 0.09, n = 8 relative current value to 0.31 ± 0.08, n = 7 post-NaHS treatment). C, co-expression with Gβγ, another allosteric enhancer of Kir3.2-PIP₂ interactions, also reduced the NaHS inhibitory effect on Kir3.2* channels (increased NaHS treated current values from 0.68 ± 0.17 n = 10 to 1.08 ± 0.20, n = 8). D, H₂S effect on Kir3.2 weakens when channel–PIP₂ interactions are strengthened through point mutations. Whereas Kir3.2 (WT) channel is greatly inhibited by NaHS treatment (0.36 ± 0.06), pore point mutation (E152D) in the background of Kir3.2 strengthens channel–PIP₂ interaction and diminishes Kir3.2 inhibition by H₂S (0.62 ± 0.36, n = 9). Additional point mutation (I234L), in the background of the Kir3.2* (E152D) mutation, further strengthens channel–PIP₂, completely abrogating the H₂S inhibition (1.04 ± 0.16, n = 8). Unpaired Student's t test was used to assess the significant differences between NaHS-untreated (control) and NaHS-treated groups, and one-way repeated-measures ANOVA and the Holm-Sidak post-test (p < 0.05) were applied to compare the NaHS-treated groups in D. Double and triple symbols p < 0.01, and p < 0.001 respectively. † indicates comparison with NaHS treated Kir3.2 group, and †† indicates comparison with NaHS-treated Kir3.2* group. Single, double, and triple symbols indicate p < 0.05, p < 0.01, and p < 0.001, respectively. N.S., non-significant.

Figure 5.

Hydrogen sulfide inhibition of Kir2.3 involves reductions in potency and efficacy of PIP₂ activation and requires micromolar concentrations of NaHS in macropatches. A, representative concentration-response experiment of NaHS in the presence of 25 μm diC8 PIP₂. B, H₂S inhibits Kir2.3 channel activity in macropatches activated submaximally by soluble PIP₂ (25 μm diC8). Direct administration of varying concentrations of NaHS to the cytoplasmic surface of inside-out patches expressing Kir2.3 channels leads to concentration-dependent inhibition of Kir2.3 channel activity (IC₅₀ = 82.95 μm and A1 value = 0.36) when they are pulsed (activated) by 25 μm soluble dioctanoyl PIP₂ (diC8). C, H₂S also inhibits Kir2.3 channel activity in macropatches maximally activated by LC-PIP₂ (full-length PIP₂), but to a lesser extent than 25 μm diC8 pulses (Fig. 3A). Direct administration of varying NaHS concentrations to the cytoplasmic surface of inside-out patches expressing Kir2.3 channels led to concentration-dependent inhibition of Kir2.3 channel activity (IC₅₀ = 73.21 μm and A1 value = 0.71) when they were activated by full-length PIP₂. D, representative experiment (summarized in E) of Kir2.3 expressing inside-out patches subject to various diC8 PIP₂ concentrations (0, 10, 25, 50, 100, 200, and 400 μm) before and after exposure to NaHS (100 μm) for 200 s. E, summary data of three experiments such as that shown in D showing that H₂S (100 μm NaHS) reduces the efficacy (increased EC₅₀ values from 6.20 μm to 9.60 μm) and potency (A2 value decreases from 1.04 to 0.74) of Kir2.3 channel activation by soluble PIP₂ (diC8).

Figure 6.

Specific cysteine residues modified in H₂S regulation of the Kir3.2 channel. A, model of sulfhydrated cysteine residues is based on Kir3.2 structure (PDB code 3SYQ), and key elements are highlighted as space-filling balls in the following colors: yellow (sulfur), red (oxygen), blue (nitrogen), and white (hydrogen). Sulfhydrated groups (sulfhydrated cysteine –SH groups) were modeled and mapped onto a structure of Kir3.2. Four cytoplasmic cysteines are highlighted: Cys-65, Cys-190, Cys-221, and Cys-321. Two extracellular cysteine residues, Cys-134 and Cys-166, located in the extracellular loop region (ECL) and involved in intersubunit disulfide interactions, were not mutated. Highlights show location of Cys-65, and how sulfhydration (space-filling models) may directly interfere with channel–PIP₂ interactions, whereas Cys-321, when sulfhydrated, may give rise to allosteric modifications at the G-loop directly as well as indirectly affect channel–PIP₂ interactions. B, Kir3.2* Cys-less mutant activity is entirely insensitive to H₂S. NaHS incubation is unable to inhibit Kir3.2*Cys-less mutant (0.985 ± 0.28, n = 23). Preliminary data reveal that the strength of Cys-less mutant–PIP₂ interaction is not significantly different from that of Kir3.2*. Re-introducing single cysteine residues to positions 65 and 321 partially restores H₂S inhibition by ∼18% (0.82 ± 0.22, n = 58) and ∼25% (0.75 ± 0.29, n = 57), respectively, roughly additive to ∼38% inhibition of Kir3.2* (0.62 ± 0.21, n = 75) with all four cytoplasmic cysteine residues. Re-introducing both cysteine residues to positions 65 and 321 simultaneously led to ∼36% inhibition of Kir3.2* (0.64 ± 0.15, n = 34), comparable with H₂S inhibition of Kir3.2* (∼38%). Re-introducing single cysteine residues to positions 190 and 221 do not significantly restore NaHS inhibition (0.91 ± 0.21, n = 11, and 0.94 ± 0.50, n = 11, respectively). Unpaired Student's t test was used to assess the significant differences between NaHS-untreated (control) and NaHS-treated groups. Non-significant (N.S.) and *** indicate comparisons with untreated groups, p < 0.001.

Figure 7.

Strength of channel–PIP₂ interactions is similar between Kir3.2* and Kir3.2* (Cys-less) channels. A, voltage protocols (in mV) used for inhibition (top) and recovery (bottom) of Kir3.2* currents by activation and deactivation of co-expressed Ci-VSP, respectively. B, Kir3.2* and Kir3.2* (Cys-less) displayed faster current inhibition compared with Kir3.2*(I234L). C, τ values for inhibition for Kir3.2* of 2.56 ± 0.147 s and Kir3.2*(Cys-less) of 2.78 ± 0.09 s were not significantly different. However, both τ values were significantly less than that of Kir3.2*(I234L) of 10.1 ± 1.76 s. One-way repeated-measures ANOVA and Holm-Sidak post-test (p < 0.05) were used to assess for statistical significance; ***, p < 0.001 compared with control. D, Kir3.2* and Kir3.2* (Cys-less) showed slower recovery time than Kir3.2* (I234L) at −80 mV. N.S., non-significant.

Figure 8.

Expression of CSE, enzymatic producer of H₂S, mimics NaHS effects. A, co-injection of CSE, integral to the production of H₂S in specific mammalian tissues at physiological concentrations, recapitulates inhibition of Kir3.2* produced by exogenously applied NaHS (2 mm) and assessed for statistical significance using one-way repeated-measures ANOVA and the Holm-Sidak post-test (p < 0.05). Increasing CSE mRNA co-injected into oocyte (with Kir3.2* channel) increases significantly Kir3.2* inhibition in a dose-dependent manner: 0.81 ± 0.12 (n = 11) at 0.5 ng injection, 0.72 ± 0.14 (n = 10) at 4.0 ng injection, 0.59 ± 0.18 (n = 11) at 8.0 ng injection. B, treatment with PAG, an inhibitor of CSE activity, reversed CSE inhibition of Kir3.2* (from 0.67 ± 0.28, n = 10, to 0.94 ± 0.40, n = 13). C, Kir3.2* Cys-less activity is refractory to CSE co-injection. Mutating all four cytoplasmic residues mentioned in Fig. 6B removes the inhibitory effect of co-expressing CSE (0.94 ± 0.26, n = 6) compared with that of Kir3.2* WT (0.48 ± 0.27, n = 10), strongly suggesting that the reduced Kir3.2* activity is not due to taxing the mRNA machinery in oocytes with a non-specific RNA load. In addition, CSE inhibition requires cytoplasmic cysteine residues as was the case with exogenously applied NaHS. Unpaired Student's t test was used to assess the significant differences between NaHS-untreated (control) and NaHS-treated groups (B) as well as between control groups and CSE co-injected oocytes. * indicates comparison with the only channel injected. † indicates comparison with co-injection with 0.5 ng of CSE. Double, and triple symbols indicate p < 0.01, and p < 0.001, respectively. N.S., non-significant.

Figure 9.

Molecular dynamics experiment provides mechanistic insight to how sulfhydration of specific residues on Kir3.2 affect channel–PIP₂ interaction and channel gating. MD simulation (25 ns) reveals how sulfhydration of Cys-65 disrupts direct channel–PIP₂ interactions, between adjacent Lys-64 and PIP₂, consequently leading to closure of the G-loop gate (decrease in distance between α carbons of Thr-317 on opposite subunits). A, left, MD simulation subjecting models of Kir3.2* (no cysteine modification) and three of the cysteine-modified, sulfhydrated Kir3.2* channels (Cys-65, Cys-221, and Cys-321) with four PIP₂ molecules for 25 ns revealed structure–dynamics–function relationship of a PIP₂-interacting residue Lys-64 (highlighted in Fig. 6A in the N-terminal region). In this simulation, the distance between Lys-64 and PIP₂ increases in the Kir3.2* model with sulfhydrated Cys-65 or Cys-321 (but not the Kir3.2* models with sulfhydrated Cys-221 or Cys-190, data not shown), suggestive of how H₂S may directly interrupt the Lys-64–PIP₂ interactions. Snapshots shown in B were selected at 17 ns (panel a), 5 ns (panel b), 15 ns (panel c), and 20 ns (panel d). B, model snapshots taken from molecular dynamics experiment details how Lys-64 residue (left, no modification) moves away from phosphate on PIP₂ head (distance between residue and PIP₂) (dotted black lines) increases from 8.1 (no sulfhydration) to 14.7 and 10.4 Å for models with Cys-65 –SH and Cys-321 –SH, respectively, whereas no substantial change is detected in the model with Cys-221 –SH. C, G-loop gate in Kir3.2* closes only when Cys-65 or Cys-321 is sulfhydrated, suggested by the decrease of distance between Thr-317 α carbons of opposite subunit G-loops (indicative of closing of G-loop gate when Cys-65 or Cys-321 is sulfhydrated). Snapshots were selected at (a) 17 ns, (b) 5 ns, (c) 15 ns, and (d) 20 ns. D, α carbons of Thr-317 residue move closer (gate closing) from no modification (gray) to when Cys-65 (red) or Cys-321(yellow) is sulfhydrated in MD simulations, while relatively no change is evident when Cys-221 (green) is sulfhydrated.

Figure 10.

Molecular dynamics experiment provides mechanistic insight as to how sulfhydration of Cys-42 is permissive of Kir6.2 channel opening. A, G-loop gate in Kir6.2 channel opened when Cys-42 was sulfhydrated, suggested by the increase in the distance between Thr-294 α carbons of the G-loops of opposite subunits. Snapshots were selected at 40 ns for both (a and b). B, top view (right) showed how the Thr-294 residue moves away (gate opening) from no modification (gray) to when Cys-42 was sulfhydrated (red).