Functionally Diverse Complement of Large Conductance Calcium- and Voltage-activated Potassium Channel (BK) α-Subunits Generated from a Single Site of Splicing

Chen, Lie; Tian, Lijun; MacDonald, Steve; McClafferty, Heather; Hammond, Martin S. L.; Huibant, Jean-Marc; Ruth, Peter; Knaus, Hans‐Guenther; Shipston, Michael J.

doi:10.1074/jbc.m505383200

Cited by 153 publications

(187 citation statements)

References 58 publications

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“…Alternative inclusion and exclusion of the seven identified splice inserts at three splice sites can form as many as 24 different splicing variants with distinct biophysical properties, or that are altered in other aspects of BK Ca channel biology. For example, expression of Strex-containing BK Ca channels is under hormonal control (46), yielding channels more sensitive to hypoxia (57), and that display a significant hyperpolarizing shift (ϳ20 mV) in V 1 ⁄2 and considerably slower rates of deactivation when compared with the insertless form (58). The Ca27 splice variant yields channels with an increased activation rate and modified Ca 2ϩ cooperativity (59).…”

Section: Discussionmentioning

confidence: 99%

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

Yan

Olsen

Park

et al. 2008

Molecular & Cellular Proteomics

101

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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 ␤ subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca 2؉ . 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 phosphoand dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BK␣ 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 BK␣ structure and function.

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Section: Discussionmentioning

confidence: 99%

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

Yan

Olsen

Park

et al. 2008

Molecular & Cellular Proteomics

101

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“…Cell surface labelling of the N-terminal Flag epitope of BK channels in nonpermeabilized HeLa cells was performed as described 45 . Briefly, coverslips with transfected cells were washed with PBS and blocked in PBS containing 3% bovine serum albumin.…”

Section: Methodsmentioning

confidence: 99%

CRL4ACRBN E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis

et al. 2014

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Ion channels regulate membrane excitation, and mutations of ion channels often cause serious neurological disorders including epilepsy. Compared with extensive analyses of channel protein structure and function, much less is known about the fine tuning of channel activity by post-translational modification. Here we report that the large conductance, Ca 2 þ -and voltage-activated K þ (BK) channels are targeted by the E3 ubiquitin ligase CRL4A CRBN for polyubiquitination and retained in the endoplasmic reticulum (ER). Inactivation of CRL4A CRBN releases deubiquitinated BK channels from the ER to the plasma membrane, leading to markedly enhanced channel activity. Mice with CRL4A CRBN mutation in the brain or treated with a CRL4A CRBN inhibitor are very sensitive to seizure induction, which can be attenuated by blocking BK channels. Finally, the mutant mice develop spontaneous epilepsy when aged. Therefore, ubiquitination of BK channels before their cell surface expression is an important step to prevent systemic neuronal excitability and epileptogenesis.

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“…The generation of full-length murine ZERO and STREX channel epitope-tagged as well as TEA-pore mutant (Y334V) constructs have been described (14,31). C-terminal ϪGFP constructs, the respective CRD constructs, and site-directed mutants were generated and sequence-verified as described in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Tian

Jeffries

McClafferty

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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105

180

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Large conductance calcium-and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the poreforming ␣-subunits. However, the molecular mechanism(s) by which phosphorylation of the ␣-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single ␣-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.L arge conductance calcium-and voltage-gated potassium (BK) channels are potently regulated by protein phosphorylation (1) and are important determinants of neuronal, cardiovascular, endocrine, and epithelial function where channel dysfunction may lead to major disorders such as hypertension (2, 3), ataxia (4), epilepsy (5, 6), and incontinence (7). BK channels are potently regulated by phosphorylation, and several putative phosphorylation motifs on the pore-forming ␣-subunit have been identified (8-12). However, as for other potassium channels, the molecular basis through which phosphorylation of the ␣-subunit effects changes in BK channel activity is essentially unknown.BK channel pore-forming ␣-subunits are encoded by a single gene, KCNMA1 (13), and native BK channels show functional heterogeneity in their response to protein kinase A (PKA)-mediated phosphorylation. This diversity results, in large part, from the extensive alternative pre-mRNA splicing of the pore-forming ␣-subunits (10, 12). Previous studies have demonstrated that PKA phosphorylation of a conserved C-terminal phosphorylation motif. RQPS 899 results in BK channel activation (9,10,14). Inclusion of ...

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Functionally Diverse Complement of Large Conductance Calcium- and Voltage-activated Potassium Channel (BK) α-Subunits Generated from a Single Site of Splicing

Cited by 153 publications

References 58 publications

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

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

CRL4ACRBN E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

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