Anionic Phospholipids Bind to and Modulate the Activity of Human TRESK Background K+ Channel
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The background K+ channel TRESK regulates sensory neuron excitability, and changes in its function/expression contribute to neuronal hyperexcitability after injury/inflammation, making it an attractive therapeutic target for pain-related disorders. Factors that change lipid bilayer composition/properties (including volatile anesthetics, chloroform, chlorpromazine, shear stress, and cell swelling/shrinkage) modify TRESK current, but despite the importance of anionic phospholipids (e.g., PIP2) in the regulation of many ion channels, it remains unknown if membrane lipids affect TRESK function. We describe that both human and rat TRESK contain potential anionic phospholipid binding sites (apbs) in the large cytoplasmic loop, but only the human channel is able to bind to multilamellar vesicles (MLVs), enriched with anionic phospholipids, suggesting an electrostatically mediated interaction. We mapped the apbs to a short stretch of 14 amino acids in the loop, located at the membrane-cytosol interface. Disruption of electrostatic lipid-TRESK interactions inhibited hTRESK currents, while subsequent application of Folch Fraction MLVs or a PIP2 analog activated hTRESK, an effect that was absent in the rat ortholog. Strikingly, channel activation by anionic phospholipids was conferred to rTRESK by replacing the equivalent rat sequence with the human apbs. Finally, in the presence of a calcineurin inhibitor, stimulation of a Gq/11-linked GPCR reduced hTRESK current, revealing a likely inhibitory effect of membrane lipid hydrolysis on hTRESK activity. This novel regulation of hTRESK by anionic phospholipids is a characteristic of the human channel that is not present in rodent orthologs. This must be considered when extrapolating results from animal models and may open the door to the development of novel channel modulators as analgesics.
KeywordsK2P channels KCNK Membrane phospholipids Nociception Neuronal excitability
Authors JPG, AC, and AA carried out cellular cultures, plasmid generation, and transfection. JPG and IE performed construction of GST fusion proteins, chimeric constructs, and liposome binding assays. AC, AA, and XG performed electrophysiological recordings in cell lines. JPG and XG participated in the design of the study and performed the statistical analysis. JPG and XG conceived the study, oversaw the research, and prepared the manuscript with help from all others. All authors read and approved the final manuscript.
Supported by grants from Ministerio de Economia y Competitividad and Instituto de Salud Carlos III of Spain FIS PI14/00141 (XG) and FIS PI17/00296 (XG), RETICs Oftared RD12/0034/0003 (XG) and RD16/0008/0014 (XG), and Generalitat de Catalunya 2014SGR1165 (XG) and 2017SGR737 (XG). J.P.G. was supported by a Ramón y Cajal Research Contract (Ministerio de Economia y Competitividad RYC-2011-08589).
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Conflict of Interest
The authors declare no competing financial interests
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