TY - JOUR

T1 - Profiling the phospho-status of the BKCa channel alpha subunit in rat brain reveals unexpected patterns and complexity

AU - Yan, Jiusheng

AU - Olsen, Jesper V

AU - Park, Kang-Sik

AU - Li, Weiyan

AU - Bildl, Wolfgang

AU - Schulte, Uwe

AU - Aldrich, Richard W

AU - Fakler, Bernd

AU - Trimmer, James S

N1 - Keywords: Algorithms; Alternative Splicing; Amino Acid Sequence; Animals; Binding Sites; Brain; Cell Line; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Phosphorylation; Potassium Channels; Protein Array Analysis; Protein Subunits; Proteomics; Rats; Recombinant Proteins; Tandem Mass Spectrometry

PY - 2008

Y1 - 2008

N2 - 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 alpha subunits (BKalpha) either alone or together with beta subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca(2+). The cytoplasmic C terminus of BKalpha 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 BKalpha 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 BKalpha differentially modulates the voltage- and Ca(2+)-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 BKalpha structure and function.

AB - 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 alpha subunits (BKalpha) either alone or together with beta subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca(2+). The cytoplasmic C terminus of BKalpha 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 BKalpha 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 BKalpha differentially modulates the voltage- and Ca(2+)-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 BKalpha structure and function.

U2 - 10.1074/mcp.M800063-MCP200

DO - 10.1074/mcp.M800063-MCP200

M3 - Journal article

C2 - 18573811

VL - 7

SP - 2188

EP - 2198

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 11

ER -