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Evidence for the interaction of Endophilin A3 with endogenous K(Ca)2.3 channels in PC12 cells

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Item Type:Article
Title:Evidence for the interaction of Endophilin A3 with endogenous K(Ca)2.3 channels in PC12 cells
Creators Name:Janbein, M., Quader, M.A., Hoppner, A.C., Grüner, I., Wanker, E., Wälter, S., Küppers, E., Grissmer, S. and Jäger, H.
Abstract:Background/Aims: Small-conductance calcium-activated (SK) channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of KCa2.3 channels and Endophilin A3 in different tissues. Endophilin A3 belongs to a family of BAR- and SH3 domain containing proteins that bind to dynamin and are involved in the process of vesicle scission in clathrin-mediated endocytosis. Methods: Using the yeast two-hybrid system and the GST pull down assay we demonstrated that Endophilin A3 interacts with the N-terminal part of KCa2.3 channels. In addition, we studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous KCa2.3 channels. KCa2.3 currents were activated by using pipette solutions containing 1 µM free Ca(2+). Results: Whole-cell measurements of PC12 cells transfected with Endophilin A3 showed a reduction of KCa2.3 specifc Cs(+) currents indicating that the interaction of Endophilin A3 with KCa2.3 channels also occurs in mammalian cells and that this interaction has functional consequences for current flowing through KCa2.3 channels. Since KCa2.3 specific currents could be increased in PC12 cells transfected with Endophilin A3 with DC-EBIO (30 µM), a known SK-channel activator, these data also implicate that Endophilin A3 did not significantly remove KCa2.3 channels from the membrane but changed the sensitivity of the channels to Ca(2+) which could be overcome by DC-EBIO. Conclusion: This interaction seems to be important for the function of KCa2.3 channels and might therefore play a significant role in situations where channel activation is pivotal for cellular function.
Keywords:K(Ca)2.3 Channel, Endophilin A3, Protein-Protein-Interaction, Yeast Two-Hybrid Experiments, Pull-Down Assays, Electrophysiology, Ca(2+) Measurements, Animals, Rats
Source:Cellular Physiology and Biochemistry
Page Range:474-490
Date:30 July 2014
Official Publication:https://doi.org/10.1159/000363016
PubMed:View item in PubMed

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