Redox Bridling of GIRK Channel Activity

pr otein-gated inw ardl y r ectifying potassium (GIRK, Kir3.x) hannels belong to the large family of inw ardl y r ectifying potasium (Kir) channels expressed throughout the body. Activation nd consequent opening of GIRK channels allow inward flow of otassium (K + ) ions into the cell resulting in membrane potenial hyperpolarization and decr eased excita bility. Thus, GIRK hannels play a key role in regulating the activity of neurons and ontrolling important physiological processes including neuonal excita bility, heart r ate , and pain per ception. 1 GIRK channels are integral membrane proteins, existing s homoor heterotetr amers. Eac h monomer features two embrane-spanning helices (M1 and M2), a re-entrant P-loop or controlling ion permeation and selectivity, and extensive ntracellular aminoand carboxy-termini crucial for channel ating. Permeation is regulated by an inner helix gate formed y the M2 segments and a cytoplasmic G-loop gate. 1 Acti v ation of GIRK channels is mediated by the direct interction of G βγ subunits, released from various G protein-coupled ece ptors (GPCRs) upon the acti v ation of inhibitory neuroransmitter r ece ptors. Howev er, the acti vity of GIRK channels epends on the presence of the membrane anionic phospholipid hosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 or PIP 2 ) while it s also modulated by ubiquitously present sodium (Na + ) ions. urthermore , GIRK c hannels ar e too r e gulated by c holesterol, hosphorylation, ethanol, etcetera. 1 The crystal structures of ecombinant GIRK channels have offered valuable insights into ow they are functionally regulated by various ligands. Thus, hannel opening is facilitated by PIP 2 at the plasma membrane, hereas G βγ and Na + modulate the c hannel’s inter action with IP 2 through conformational changes that govern the gating proess. 2 The intracellular milieu is a reducing environment charcterized by a balanced redox state. This state is crucial to upport cellular processes while serving as a pr otecti v e shield

G pr otein-gated inw ardl y r ectifying potassium (GIRK, Kir3.x) channels belong to the large family of inw ardl y r ectifying potassium (Kir) channels expressed throughout the body. Activation and consequent opening of GIRK channels allow inward flow of potassium (K + ) ions into the cell resulting in membrane potential hyperpolarization and decr eased excita bility. Thus, GIRK channels play a key role in regulating the activity of neurons and controlling important physiological processes including neur onal excita bility, heart r ate , and pain per ception. 1 GIRK channels are integral membrane proteins, existing as homo-or heterotetr amers. Eac h monomer features two membrane-spanning helices (M1 and M2), a re-entrant P-loop for controlling ion permeation and selectivity, and extensive intracellular amino-and carboxy-termini crucial for channel gating. Permeation is regulated by an inner helix gate formed by the M2 segments and a cytoplasmic G-loop gate. 1 Acti v ation of GIRK channels is mediated by the direct interaction of G βγ subunits, released from various G protein-coupled r ece ptors (GPCRs) upon the acti v ation of inhibitory neurotransmitter r ece ptors. Howev er, the acti vity of GIRK channels depends on the presence of the membrane anionic phospholipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 or PIP 2 ) while it is also modulated by ubiquitously present sodium (Na + ) ions. Furthermore , GIRK c hannels ar e too r e gulated by c holesterol, phosphorylation, ethanol, etcetera. 1 The crystal structures of recombinant GIRK channels have offered valuable insights into how they are functionally regulated by various ligands. Thus, channel opening is facilitated by PIP 2 at the plasma membrane, whereas G βγ and Na + modulate the c hannel's inter action with PIP 2 through conformational changes that govern the gating process. 2 The intracellular milieu is a reducing environment characterized by a balanced redox state. This state is crucial to support cellular processes while serving as a pr otecti v e shield a gainst dama ging r eacti v e oxygen species (R OS) ther eby facilitating enzymatic reactions and energy production. 3 GIRK channels exhibit low basal activity in reducing intracellular environments. Nevertheless, the channel's behavior in nati v e tissues is influenced by a variety of cellular factors, which complicate the interpretation of their specific contributions to channel gating.
In a recent Function publication, Lee et al. effectively bypassed the complexity of cellular systems to study the regulation of GIRK channels. 4 The team employed an acellular in vitro flux assay to manipulate the redox state of the functional channel protein within liposomes made of determined phospholipid mixtur es. Subsequentl y, they assessed GIRK2-mediated fluxes upon systematic r e placement of key modulatory co-factors. This method successfully reproduced the channel's typical behavior (ie, low basal activity in reducing conditions) while also uncovering an oxidation-dependent anomalous enhancement of GIRK2 acti v ation.
It w as observ ed that in the a bsence of stringent r edox control, as is commonly provided b y health y cells, the GIRK2 protein undergoes oxidation at two crucial cysteine residues during purification. These cysteines have different susceptibility to environmental oxidation, and their oxidized state appears to play a pi v otal r ole in GIRK2 gating. Two distinct effects were observed upon oxidation: (i) the loss of PIP 2 and Na + -dependent activity, linked to cysteine 65 and (ii) a slower rise in basal activity associated with cysteine 190.
The N-terminal cysteine at position 65 of GIRK2 is highly conserved in the Kir channel family and it is str ate gically located adjacent to lysine 64, which contributes to the PIP 2 binding site. Mor eov er, C65 is positioned tow ard a neighboring subunit's poc ket, whic h is inv olv ed in the Na + binding site (Figures 5B and 7A in Lee et al. 4 ). Nota b l y, C65 plays a role in GIRK current inhibition by Cd 2 + through its direct interaction with a high-affinity locus at the entrance of the Na + binding site. 5 Cysteine 190, a conserv ed r esidue among the Kir3 and Kir6 subfamilies, is positioned at the cytoplasmic end of the TM2 inner helix near the gate residue phenylalanine 192. It faces 1 outwar d, to war d the outer helix TM1, lipid membr ane , and the phosphatidylinositol molecule tail. Unlike the cytoplasmically exposed C65, C190 appears buried within the channel protein consistent with the slower time course of oxidation r ev ealed by the study.
Lee et al. 4 unco ver ho w the protein purification environment, especially harsh oxidizing conditions, affects GIRK2 channel function. They examined different GIRK2 structures with PIP 2 , Na + , and G βγ , emphasizing slight rearrangements near the neighboring regions of C65 and C190. This structural comparison provides evidence that modifications to these cysteines may impact the channel's gating mechanism. The conservation of specific oxidiza b le cysteine residues across multiple species in GIRK channels suggests that oxidation-mediated regulation may influence their physiological function in various organisms.
Recombinant GIRK channels expressed in Xenopus laevis oocytes have been shown to display r edox-de pendent gating mediated through cytoplasmic cysteine residues. [6][7][8] However, experiments conducted on homo-and heteromeric GIRK channels in cellular systems have yielded conflicting results. Specifically, GIRK channels exhibited increased activity upon application of ROS 8 and the reducing agent DTT. 6 Gi v en the appar ent impact of pr otein oxidation on GIRK channel function, investigating the potential influence of lipid peroxidation on membrane/liposome composition and GIRK channel behavior could yield v alua b le insight.
When there is an imbalance between ROS and antioxidants, free r adicals gener ated during cell metabolism trigger oxidati v e str ess. This str ess dama ges pr oteins, lipids, and DNA, disrupting cellular homeostasis. Hence, it is implicated in various physiological and pathophysiological processes. Mounting evidence has implicated GIRK channel dysfunction in the pathophysiology of neurological and car dio vascular disor ders. 9 , 10 Ho wev er, the pr ecise mechanisms by which GIRK channels are involved in these diseases remain incompletely understood.
The recent findings from the Nichols' lab, combined with pr evious r esear c h on cellular systems, highlight the oxidati v e state as a crucial factor influencing GIRK function. This could hold significant r elev ance in scenarios where heightened meta bolic acti vity, such as during e pisodes of neur onal firing and rapid cardiac myocyte contractions, overlaps with pathological conditions c har acterized by incr eased pr oduction of ROS or impaired antioxidant capacity. Considering the influence of ROS on the activity of ion channels and transporters, unbridled acti v ation of GIRK channels may serv e as a critical link connecting cellular metabolic state, ionic homeostasis, and excitability.

Funding
R.K.F-U. is supported by the Australian Research Council's Discov er y Pr oject (DP210102405).