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.
Highlights
Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells
We have identified extensive in vivo phosphorylation of native BK␣ and examined the functional consequence of phosphorylation at a number of identified sites by electrophysiological analyses of phospho- and dephosphomimetic BK␣ mutants transiently expressed in HEK-293 cells
MS Analyses of Rat Brain BK␣: Primary Sequence Coverage and Splice Variations—Two independent proteomic analyses of BK␣ affinity-purified from membrane preparations of total rat brain form the basis of this study
Summary
Affinity Purification of BKCa Channels from Rat Brain—Two different sets of affinity purifications of BKCa channels were used in this study. Database searches were performed with a peptide mass tolerance of 2 Da (LTQ) or 20 ppm (LTQ-FT), MS/MS tolerance of 0.4 Da, and strict tryptic specificity (cleavage after lysine and arginine) allowing one missed cleavage site; carbamidomethylation of Cys was set as fixed modification, whereas oxidation (Met), N-acetylation, N-pyroglutamine formation, and phosphorylation (Ser, Thr, Tyr) were considered as variable modifications. The peptide mixtures from BK␣ affinity-purified with the polyclonal antibodies anti-BK␣_2 and anti-BK␣_3 were separated by online high pressure liquid chromatography and directly electrosprayed into an LTQ-Orbitrap hybrid mass spectrometer as described [41]. Inside-out patches were continuously perfused with internal solution using a sewer pipe flow system (DAD-12; Adams and List Assoc., Ltd.); computer-controlled switches allowed for complete solution exchange at excised patches in Ͻ1 s
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