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 (PIP 2 ) 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-PIP 2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H 2 S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide (NaHS) as an exogenous H 2 S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H 2 S 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 H 2 S regulation depended on the strength of the channel-PIP 2 interactions. H 2 S regulation was attenuated when channel-PIP 2 interactions were strengthened and was increased when channel-PIP 2 interactions were weakened by depleting PIP 2 levels. These H 2 S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H 2 S inhibition and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteinelacking mutant rescued H 2 S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP 2 interactions and channel gating.The gaseous mediator, hydrogen sulfide (H 2 S), is increasingly garnering the reputation of a major physiological messenger molecule with robust effects in ischemia-related insults to the heart, brain, and other peripheral tissues (1). H 2 S is generated from L-cysteine by three distinct enzymes: cystathionine b-synthase (CBS), 2), and signals via sulfhydration (also known as persulfidation), a post-translational modification of reactive cysteine residues analogous to Nnitrosylation by nitric oxide (3). In these studies, sulfhydration was detected by techniques such as a Biotin Switch Assay (4), a Tag Switch Assay (TSA) (5), as well as mass spectrometry (6). In experiments in-vivo and in-vitro, the endogenous production of H 2 S was modulated by several chemicals and/or H 2 S was applied exogenously to tissue/cells, using sulfide salts, mainly sodium hydrosulfide (NaHS). Collectively, H 2 S has been shown to play a role in diverse physiological processes, such as cellular necrosis, apoptosis, oxidative stress, or inflammation (7). One theme emerging from these studies is that H 2 S increases the excitability of neuronal compartments (8-12), whereas it depresses excitability of cardiac tissues (13,14), correlating to neurotoxic (12,15) and cardioprotective (16)(17)(18)(19)(20) effe...

Oxidation driven reversal of PIP2-dependent gating in GIRK2 channels

“…That these two consequences result from distinct oxidation events is suggested by their temporal separation: agonist activation is lost within 30 minutes, but increase of basal activity takes a few hours under our experimental conditions. The C65V mutation completely abolished PIP 2 activation, even under fully reduced c o n d i t i o n s. This implicates C65 oxidation as being responsible for the rapid loss of agonist-activation, and is consistent with previous studies that reported loss of cellular GIRK2 activity when this cysteine residue is mutated to various other amino acids or otherwise modified 19,22,[45][46][47] . The molecular mechanism for the loss of agonist activation is yet to be determined; the mutation may abolish PIP 2 binding or prevent conformational change required for PIP 2 binding, or for transduction from binding to channel opening.…”

Gas Signaling Molecules and Mitochondrial Potassium Channels

“…It is possible that the S-sulfhydration of Cys43 in Kir6.1 impedes the binding of ATP to the channel due to electrostatic or spatial changes, which could result, in turn, in increased access of PIP 2 to its binding site [ 127 ]. It was recently shown that H 2 S affects the activity of other Kir channels, Kir2 and Kir3, in a manner opposite to that of Kir6.1 mentioned above [ 128 ]. Specifically, the use of NaHS as an H 2 S donor resulted in the inhibition of Kir3.2 channels in Xenopus oocytes and Kir3.2 channels in CHO-K1 cells.…”

Mitochondrial mechanisms by which gasotransmitters (H2S, NO and CO) protect cardiovascular system against hypoxia

“…Herewith, the enhancement in the activity of K AT P channels was accompanied with an increase of the conjugation of PIP 2 with the corresponding Kir6.1 sites that stabilized the channel in the open state and led to an increase in the amplitude of the K + current [61]. It has been recently shown that H 2 S changes activity of other isoforms of Kir subunits (Kir2 and Kir3) of K AT P channels [62] that may also contribute to cardioprotection [63,64].…”