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.
Highlights
Kv2.1 channels are components of plasma membrane:endoplasmic reticulum membrane contact sites (PM:ER MCS)
The cells with clustered Kv2.1 typically had condensed chromosomes as revealed with the Hoechst dye (Fig. 1A). We confirmed that these robust differences in Kv2.1 localization in different subpopulations within the same cultures were related to the cell cycle stage by immunolabeling for histone H3 phosphorylated at Ser-10 (H3pS10; Fig. 1A), an M phase marker [57]
A significantly increased incidence of cells with clustered Kv2.1 was observed in M phase cells regardless of whether the cell cycle stage was determined by Hoechst staining and the presence or absence of condensed chromosomes, or by H3pS10 immunolabeling (Fig. 1D)
Summary
Kv2.1 channels are components of plasma membrane:endoplasmic reticulum membrane contact sites (PM:ER MCS). Kv2.1 is present in large PM clusters (2, 8 –10), which are located at sites overlaying subsurface cisternae [11], which in certain neurons contain high levels of ryanodine receptor intracellular Ca2ϩ release channels [12,13,14] These sites represent specialized PM:endoplasmic reticulum (ER) membrane contact sites (i.e. PM:ER MCS [15]). The clustering, phosphorylation state, and association with PM:ER MCS of Kv2.1 during mitosis, when robust changes in membrane structure throughout the cell are driven by cell cycle-dependent changes in protein kinase and phosphatase activity [30] leading to widespread changes in cellular protein phosphorylation [31], has not been investigated. Our findings on cell cycle-dependent phosphorylation and clustering of Kv2.1 suggest that changes in the subcellular compartmentalization of PM proteins, including those associated with PM:ER MCS, may be a more common feature of cell cycle-dependent changes in cell structure than previously recognized
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