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Molecular & Cellular Proteomics 7:2188-2198, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Profiling the Phospho-status of the BK_Ca Channel Subunit in Rat Brain Reveals Unexpected Patterns and Complexity^*,S

Jiusheng Yan^,, Jesper V. Olsen^¶, Kang-Sik Park, Weiyan Li, Wolfgang Bildl^||, Uwe Schulte^**, Richard W. Aldrich, Bernd Fakler^||, and James S. Trimmer^,,¶¶

From the Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, California 95616, Section of Neurobiology, School of Biological Sciences, University of Texas, Austin, Texas 78712, ^¶ Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany, ^|| Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany, ^** Logopharm GmbH, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany, Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, California 95616

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BK_Ca) channels are tetramers of subunits (BK) either alone or together with β subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca²⁺. The cytoplasmic C terminus of BK is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BK_Ca channels affinity-purified from rat brain to analyze in vivo BK phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BK differentially modulates the voltage- and Ca²⁺-dependence of channel activation. These results demonstrate that the pore-forming subunit of BK_Ca channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BK structure and function.

^¶¶ To whom correspondence may be addressed: Section of Neurobiology, Physiology and Behavior, College of Biological Sciences, 196 Briggs Hall, University of California, One Shields Ave., Davis, CA 95616-8519. E-mail: jtrimmer{at}ucdavis.edu

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