MaxiK channel interactome reveals its interaction with GABA transporter 3 and heat shock protein 60 in the mammalian brain. (11th March 2016)

• Record Type: Journal Article
• Authors: Singh, H.; Li, M.; Hall, L.; Chen, S.; Sukur, S.; Lu, R.; Caputo, A.; Meredith, A.L.; Stefani, E.; Toro, L.
• Is Part Of: Neuroscience. Volume 317(2016)
• Page Start: 76
• Page End: 107
• Publication Date: 2016-03-11
• Subjects: CP-AMPARs calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors -- EDTA ethylenediaminetetraacetic acid -- FBS fetal bovine serum -- FLAG DYKDDDDK -- GABA γ-aminobutyric acid -- GAT3 GABA transporter 3 -- HEK293T human embryonic kidney 293 cells containing SV40 T-antigen -- HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid -- HSP60 heat shock protein 60 kDa 1 -- IgG immunoglobulin G -- LC–MS/MS liquid chromatography–tandem mass spectrometry -- mAb monoclonal antibody -- MaxiK (Kcnma1) large conductance calcium and voltage activated potassium channel -- NGS normal goat serum -- pAb polyclonal antibody -- PBS phosphate-buffered saline -- PPI protein proximity index -- SDS–PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis -- TIM translocase of the inner membrane -- TOM translocase of the outer membrane -- Tris tris(hydroxymethyl)aminomethane; MaxiK interactome -- BKCa -- proteomics -- mitochondria -- GABA transporter -- HSP60; Neurochemistry -- Periodicals; Neurophysiology -- Periodicals; Neurology -- Periodicals; Neurochimie -- Périodiques; Neurophysiologie -- Périodiques; Neurochemistry; Neurophysiology; Electronic journals; Periodicals; Electronic journals; 612.8
• DOI: 10.1016/j.neuroscience.2015.12.058
• Languages: English
• ISSNs: 0306-4522

Abstract

Highlights: Identified the interactome of MaxiK channels in the mouse brain containing 1185 proteins using two independent approaches. Established localization of MaxiK channels in the brain mitochondria. Confirmed interaction of MaxiK channel with heat shock protein 60. Confirmed interaction of MaxiK channel with GAT3. Abstract: Large conductance voltage and calcium-activated potassium (MaxiK) channels are activated by membrane depolarization and elevated cytosolic Ca 2+ . In the brain, they localize to neurons and astrocytes, where they play roles such as resetting the membrane potential during an action potential, neurotransmitter release, and neurovascular coupling. MaxiK channels are known to associate with several modulatory proteins and accessory subunits, and each of these interactions can have distinct physiological consequences. To uncover new players in MaxiK channel brain physiology, we applied a directed proteomic approach and obtained MaxiK channel pore-forming α subunit brain interactome using specific antibodies. Controls included immunoprecipitations with rabbit immunoglobulin G (IgG) and with anti-MaxiK antibodies in wild type and MaxiK channel knockout mice ( Kcnma1 −/− ), respectively. We have found known and unreported interactive partners that localize to the plasma membrane, extracellular space, cytosol and intracellular organelles including mitochondria, nucleus, endoplasmic reticulum and Golgi apparatus. Localization of MaxiK channel to mitochondria was further confirmed using purified brain mitochondria colabeled with MitoTracker. Independent proof of MaxiK channel interaction with previously unidentified partners is given for GABA transporter 3 (GAT3) and heat shock protein 60 (HSP60). In human embryonic kidney 293 cells containing SV40 T-antigen (HEK293T) cells, both GAT3 and HSP60 coimmunoprecipitated and colocalized with MaxiK channel; colabeling was observed mainly at the cell periphery with GAT3 and intracellularly with HSP60 with protein proximity indices of ∼0.6 and ∼0.4, respectively. In rat primary hippocampal neurons, colocalization index was identical for GAT3 (∼0.6) and slightly higher for HSP60 (∼0.5) association with MaxiK channel. The results of this study provide a complete interactome of MaxiK channel the mouse brain, further establish the localization of MaxiK channel in the mouse brain mitochondria and demonstrate the interaction of MaxiK channel with GAT3 and HSP60 in neurons. The interaction of MaxiK channel with GAT3 opens the possibility of a role of MaxiK channel in GABA homeostasis and signaling.