title: Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels.
abstract: All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81-104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404-404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification.
authors.affiliation: From the Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, the Jiangsu Province Key Laboratory of Anesthesiology and Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, XuZhou Medical College, Xuzhou, 221004 Jiangsu Province, China.
authors.forename: Qiong-Yao
authors.initials: QY
authors.lastname: Tang
authors.affiliation: the Department of Biology, University of Richmond, Richmond, Virginia 23173.
authors.forename: Trevor
authors.initials: T
authors.lastname: Larry
authors.affiliation: the Department of Biology, University of Richmond, Richmond, Virginia 23173.
authors.forename: Kalen
authors.initials: K
authors.lastname: Hendra
authors.affiliation: the Department of Biology, University of Richmond, Richmond, Virginia 23173.
authors.forename: Erica
authors.initials: E
authors.lastname: Yamamoto
authors.affiliation: the Department of Chemistry and Biochemistry, University of San Diego, San Diego, California 92110, and.
authors.forename: Jessica
authors.initials: J
authors.lastname: Bell
authors.affiliation: From the Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298.
authors.forename: Meng
authors.initials: M
authors.lastname: Cui
authors.affiliation: From the Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298.
authors.forename: Diomedes E
authors.initials: DE
authors.lastname: Logothetis
authors.affiliation: the Department of Biology, University of Richmond, Richmond, Virginia 23173 lboland@richmond.edu.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Boland
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D019143:Evolution, Molecular; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D011161:Porifera / Q000235:genetics / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014714:Vertebrates / Q000145:classification / Q000235:genetics* / Q000378:metabolism
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D024661:Potassium Channels, Inwardly Rectifying
keywords: IRK channel; TEVC; evolution; gating; inositol phospholipid; inwardly rectifying; molecular modeling; patch clamp; phosphatase; sponge