Human EAG Channels Are Modulated By Phospholipids

Ion channels allow transmembrane fluxes of specific ions,which constitutes the molecular basis of cell electrical activity and plays major roles in both physiological activities and pathophysiological processes.Aberrant ion channel functions contribute to canonical diseases such as epilepsy,cardiac arrhythmia,and neuromuscular symptoms collectively known as channelopathies.Increased evidence suggests that cancer constitutes another category of channelopathies.Indeed,some ion channel modulators have demonstrated antitumor efficacy.Given the cell surface localization and moderately specific strucuture properties,pharmacological strategies to target ion channel could prove to be promising channelopathy therapeutics.In fact,around13%of currently known drugs whose primary therapeutic targets are ion channels.Among the large family of ion channels,potassium channels represent the most diverse group,in terms of both their biochemical structures and their roles in channelopathies,they might be the channels for which the noval targeted therapies could be identified.One of most attractive potassium channel is human voltage-gated etheràgo-go?hEAG1?K⁺channel because its aberrant overactivation is associated with over 70%cancer and some neuronal disorders such as Zimmermann-Laband and Temple-Baraitser syndromes.Thus,hEAG1channels are been considered as a tumor marker as well as a potential therapeutic target for both cancers and neuronal disorders.Even so,the regulation of hEAG1 functional properties is only poorly understood because only a few endogenous regulators of the hEAG1 channels have been identified.Thus,focusing on identifying novel endogenous regulators of hEAG1 channels is necessary in order to understand their physiological roles and design therapeutic agents for cancer and neuronal disorders.The functional regulation of plasma membrane proteins by lipid molecules is an integral component of cell function and metabolism.As a most abuntdant variant of inositide lipids in the inner leaflet of membranes,PIP₂ is the phospholipid precursor of the second messagers inositol trisphosphate?IP₃?,diacylglycerol?DAG?and itself is known to be a necessary cofactor for a variety of ion channels,such as Kv,Kir,KCNQ and Cav channels.PIP₂ plays a vital part in cellular signaling by directly or indirectly interacting with ion channels.The potential importance of PIP₂on functional regulation of hEAG channels has not been reporeted,which prompted us to investigate whether hEAG1 channels is regulated by PIP₂.By inside-out and whole-cell patch-clamp recordings,our results showed that brain-derived PIP₂ rapidly decreased the steady-state current size.Depletion of endogenous PIP₂ either by serotonin-induced phospholipase C?PLC?activation or by a rapamycin-induced translocation system enhanced the channel activity at physiological membrane potentials,suggesting that PIP₂ exerts a tonic inhibitory influence.The inhibitory effect was concentration dependent and dependent on the length of the fatty acid chains and negative charges of the head group.To test whether PIP₂ directly interacts with the hEAG1 channel,we used bio-layer interferometry?BLI?,a novel methodology that has emerged recently for detecting protein-protein interactions as well as those between proteins and small molecules to measure the kinetics of binding of PIP₂ to the purified hEAG1 channel complex.The BLI measurements showed the existence of two kinetic components in association and dissociation.The kinetics of the BLI signal resembled those observed in the patch-clamp measurements in the excised patches.The close correspondence between the BLI and electrophysiological results demonstrated a direct interaction between PIP₂ and hEAG1 channel protein.Furthermore,our study,combining electrophysiological,mutagenesis and BLI assays identifies a short N-terminal segment of the channel necessary for PIP₂ binding.In addition,the putative PIP₂ interaction sites overlap with CaM binding sites,but they are functional independence in the regulation of hEAG1channels.In addition to hEAG1,we also found that PIP₂ significantly inhibited hEAG2?KCNH5,Kv10.2?channels,which have virtually the same sequence near the CaM-N area as hEAG1.Based on the gating mechanisms obtasined from the crystal structure of eag-CNBHD complex formed by the eag domain in the N terminus and the C-terminal CNBHD,we propose that the potential conformational aleteration in eag-CNBHD complex is involved in PIP₂ induced hEAG1 channel gating inhibition.Based on our data,we could therefore draw the follwing conclusions:?i?Inhibition of hEAG1 channels by exogenous and endogenous PIP₂;?ii?PIP₂ directly binds to hEAG1 channels;?iii?PIP₂inhihits hEAG1 by binding to a short N-terminal segment of the channel;?iv?Functional independence between PIP₂ and Ca²⁺/CaM in the regulation of hEAG1 channels.In summary,we identifies PIP₂ as a noval inhibitory gating modifier of hEAG1 channels,suggesting that manipulations of the PIP₂ signaling pathway may represent a strategy to treat hEAG1 channel-associated diseases.The main innovation points of this research are as follows:?i?In this thesis,we show,for the first time,that the tonic hEAG1 channel is inhibited by PIP₂,and identify PIP₂ as an endogenous regulator of hEAG1channel.Thus,our finding that PIP₂ is a potent inhibitory gating modifier of hEAG1 channels is expected to provide considerable insight into the pathology of the hEAG1 channel-associated diseases and offer new therapeutic pathways in their treatment.?ii?We firstly used BLI technology to analyze the interaction between ion channel and small molecule in our research.Our success application of this noval method suggests a model for investigating other channels interaction with proteins or small molecule.