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. 2008 Nov;7(11):2188-98.
doi: 10.1074/mcp.M800063-MCP200. Epub 2008 Jun 23.

Profiling the Phospho-Status of the BKCa Channel Alpha Subunit in Rat Brain Reveals Unexpected Patterns and Complexity

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Free PMC article

Profiling the Phospho-Status of the BKCa Channel Alpha Subunit in Rat Brain Reveals Unexpected Patterns and Complexity

Jiusheng Yan et al. Mol Cell Proteomics. .
Free PMC article

Abstract

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.

Figures

F<sc>ig</sc>. 1.
Fig. 1.
Affinity purification of BKα from plasma membrane-enriched protein fractions prepared from total rat brain. A, SDS-PAGE separation of eluates from affinity purifications with three antibodies targeting different epitopes in the C terminus of BKα. The gel was silver-stained; arrowhead denotes the band containing BKα. B, immunoblot of input (appropriately solubilized membrane preparations), flow through (unbound), and eluate of affinity purifications of BKα with the indicated antibodies after SDS-PAGE separation. The source and unbound lanes were loaded with samples normalized to equivalent amount of raw material. Note the complete depletion of the source material of BKα.
F<sc>ig</sc>. 2.
Fig. 2.
In vivo phosphorylation sites and splice inserts of rat brain BKα identified by MS analysis. A, amino acid sequence of the constitutive form of BKα (Swiss-Prot accession number Q62976–2; last 8 residues from splice insert are not included) together with the identified phospho-Ser/Thr residues (highlighted in blue) and the splice insertion sites (marked by colored triangles). Dot-free Ser/Thr residues denote unambiguous phosphorylation sites, whereas dots underneath Ser/Thr residues mark ambiguous sites. B, amino acid sequence of the identified N-terminal extension and splice variants. Peptides identified by MS analysis are shown in red, those not identified in MS analyses are in gray. Name of each splice insert is given in bracket on the right side of the amino acid sequence. Horizontal bars denote hydrophobic segments S0–S10.
F<sc>ig</sc>. 3.
Fig. 3.
Representative MS/MS spectra of two phosphopeptides harboring either one phosphothreonine (Thr(P)-965, upper panel) or three phosphoserines (Ser(P)-854/Ser(P)-855/Ser(P)-859, lower panel). Spectra were obtained on an LTQ-Orbitrap mass spectrometer with multistage activation (pseudo-MS3) of the precursor ions shown in the insets.
F<sc>ig</sc>. 4.
Fig. 4.
Localization of identified phosphosites and splice variants on rat brain BKα. Membrane topology of BKα together with localization of the identified phosphosites and splice insertions. The constitutive form of BKα contains the transmembrane core (S0–S6), the pore region, the hydrophobic intracellular segments S7–S10, the Ca2+ bowl, and the RCK domains (25, 65, 66). Insertion sites of sequence stretches generated by alternative splicing or alternative start of translation are indicated by triangles with the same color coding and names as in Fig. 2. Phosphosite Ser(P)-855 exhibiting a marked effect on channel gating is highlighted in red.
F<sc>ig</sc>. 5.
Fig. 5.
Functional characterization of the Ser-855 phosphorylation site. Macroscopic currents recorded in inside-out patches excised from HEK-293 cells transiently expressing wild type or mutant (S855A and S855D) BKCa channels. A, representative current traces through WT channels recorded at the indicated values for [Ca2+]i, current scaling is 1 nA, time scaling as indicated. B, activation curves of WT, S855A, and S855D channels. Lines represent fits of single Boltzmann functions to the data points (mean ± S.E., numbers given in Table I).
F<sc>ig</sc>. 6.
Fig. 6.
Comparison of MS-identified phosphosites on BKα with sites predicted by computer algorithms. A, phosphorylation site predictions for all Ser/Thr residues of BKα (including splice variations) by the indicated algorithms. Scores (given as relative values) considered significant by the individual algorithms are marked by the gray bar and by non-shaded symbols (for predictions by Scansite). Phosphorylation sites predicted by PhosphoSite are given as filled green circles; MS-identified sites are denoted by black (unambiguous sites) and gray rectangles (ambiguous sites). Ser/Thr residues inaccessible to our MS analyses are marked by horizontal dashes. B, overlap of phosphosites as predicted by at least three or four computer algorithms (left and middle panel; for details on the 7 algorithms used see Supplemental Material) or by phosphosite with the 30 sites identified by the MS analyses presented here. Predicted sites are depicted as blue circles, identified sites by red circles.

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