Hydrogen Sulfide (H2S) Regulation of Kir (Inwardly Rectifying K+) Channels

Abstract

Inwardly rectifying potassium channels establish and regulate the resting membrane potential of excitable cells in tissues such as brain and heart. A wealth of electrophysiological, structural, and biochemical studies has revealed that a biological phospholipid, Phosphatidylinositol 4,5-bisphosphate(PIP2), is a key direct activator of Kir(Inwardly-rectifying K+) channels. Here we show that hydrogen sulfide(H2S), an emerging gasotransmitter, produced in cells, regulates the activity of several members of the Kir channel family differentially via s-sulfhydration of its cytoplasmic cysteine residues and the extent of regulation depends on the strength of channel-PIP2 interaction. These findings have used two independent methods to demonstrate H2S effects: direct administration of an exogenous donor, sodium hydrogen sulfide(NaHS), and expression of cystathionine gamma lyase(CSE), the principle producer of endogenous H2S in peripheral tissues, such as in the cardiovascular system and specific regions of the brain. We show here that H2S regulation depends on the strength of channel-PIP2 interactions by employing techniques that alter the extent of channel-PIP2 interactions. H2S regulation is attenuated when strengthening channel-PIP2 interactions either through point mutations in Kir3.2(altering channel structure intrinsically), interacting proteins(e.g. G-beta-gamma) or by increasing intracellular PIP2 levels by co-expression of PIP5Kinase(PIP5K), the enzyme that converts endogenous PIP(4) into PIP2(4,5). Conversely, our results show increased H2S regulation when channel-PIP2 interactions are weakened through the administration of Wortmannin(Wort), a known blocker of PIP4K, which leads to abatement of intracellular PIP2. In addition, activation of co-expressed Ci-VSP, a voltage activated PIP5 phosphatase, also reduces intracellular PIP2, and H2S is able to exert greater effects on the channel. These H2S effects depend on the presence of specific cytoplasmic cysteine residues in Kir3.2(GIRK2), as their replacement(by other non-polar amino acids) abolishes the H2S effect(without affecting channel-PIP2 interactions). Reintroduction of specific cysteine residues back in to the background of this GIRK2 cysteine less mutant rescues the H2S effect. In order to monitor H2S modification, we employ the biochemical technique, Tag Switch Assay(TSA), showing that Kir3.2 is directly s-sulfhydrated at specific cysteine residues upon addition of exogenous H2S via administration of NaHS to wild-type Kir3.2 and Kir3.2 mutants lacking specific cysteine residues. Lastly, molecular dynamics simulation experiments are providing mechanistic insight in how s-sulfhydration of these specific cysteine residues would lead to changes in affecting channel-PIP2 interactions and channel gating. These studies reveal for the first time the intricate interplay between Kir channels(proteins), PIP2(biological lipids) and H2S(gaseous molecules).