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

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 α subunits (BKα) 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 BKα 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 BKα 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 BKα 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 BKα structure and function.