title: Cell Cycle-dependent Changes in Localization and Phosphorylation of the Plasma Membrane Kv2.1 K+ Channel Impact Endoplasmic Reticulum Membrane Contact Sites in COS-1 Cells.
abstract: The plasma membrane (PM) comprises distinct subcellular domains with diverse functions that need to be dynamically coordinated with intracellular events, one of the most impactful being mitosis. The Kv2.1 voltage-gated potassium channel is conditionally localized to large PM clusters that represent specialized PM:endoplasmic reticulum membrane contact sites (PM:ER MCS), and overexpression of Kv2.1 induces more exuberant PM:ER MCS in neurons and in certain heterologous cell types. Localization of Kv2.1 at these contact sites is dynamically regulated by changes in phosphorylation at one or more sites located on its large cytoplasmic C terminus. Here, we show that Kv2.1 expressed in COS-1 cells undergoes dramatic cell cycle-dependent changes in its PM localization, having diffuse localization in interphase cells, and robust clustering during M phase. The mitosis-specific clusters of Kv2.1 are localized to PM:ER MCS, and M phase clustering of Kv2.1 induces more extensive PM:ER MCS. These cell cycle-dependent changes in Kv2.1 localization and the induction of PM:ER MCS are accompanied by increased mitotic Kv2.1 phosphorylation at several C-terminal phosphorylation sites. Phosphorylation of exogenously expressed Kv2.1 is significantly increased upon metaphase arrest in COS-1 and CHO cells, and in a pancreatic β cell line that express endogenous Kv2.1. The M phase clustering of Kv2.1 at PM:ER MCS in COS-1 cells requires the same C-terminal targeting motif needed for conditional Kv2.1 clustering in neurons. The cell cycle-dependent changes in localization and phosphorylation of Kv2.1 were not accompanied by changes in the electrophysiological properties of Kv2.1 expressed in CHO cells. Together, these results provide novel insights into the cell cycle-dependent changes in PM protein localization and phosphorylation.
authors.affiliation: From the Departments of Neurobiology, Physiology, and Behavior.
authors.forename: Melanie M
authors.initials: MM
authors.lastname: Cobb
authors.affiliation: Physiology and Membrane Biology, and.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Austin
authors.affiliation: Physiology and Membrane Biology, and Anesthesiology and Pain Medicine, University of California Davis School of Medicine, Davis, California 95616.
authors.forename: Jon T
authors.initials: JT
authors.lastname: Sack
authors.affiliation: From the Departments of Neurobiology, Physiology, and Behavior, Physiology and Membrane Biology, and jtrimmer@ucdavis.edu.
authors.forename: James S
authors.initials: JS
authors.lastname: Trimmer
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002453:Cell Cycle; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D051669:Shab Potassium Channels / Q000378:metabolism*
chemical_list: C496991:Kcnb1 protein, rat; D051669:Shab Potassium Channels
keywords: endoplasmic reticulum (ER); membrane structure; mitosis; plasma membrane; potassium channel; protein phosphorylation; subcellular organelle