ABSTRACT
Cellular responses to hypoxia are tissue-specific and dynamic. However, the mechanisms that underlie this differential sensitivity to hypoxia are unknown. Large conductance voltage- and Ca-activated K (BK) channels are important mediators of hypoxia responses in many systems. Although BK channels are ubiquitously expressed, alternative pre-mRNA splicing of the single gene encoding their pore-forming alpha-subunits provides a powerful mechanism for generating functional diversity. Here, we demonstrate that the hypoxia sensitivity of BK channel alpha-subunits is splice-variant-specific. Sensitivity to hypoxia is conferred by a highly conserved motif within an alternatively spliced cysteine-rich insert, the stress-regulated exon (STREX), within the intracellular C terminus of the channel. Hypoxic inhibition of the STREX variant is Ca-sensitive and reversible, and it rapidly follows the change in oxygen tension by means of a mechanism that is independent of redox or CO regulation. Hypoxia sensitivity was abolished by mutation of the serine (S24) residue within the STREX insert. Because STREX splice-variant expression is tissue-specific and dynamically controlled, alternative splicing of BK channels provides a mechanism to control the plasticity of cellular responses to hypoxia.
Subject(s)
Cell Hypoxia/physiology , Cysteine/metabolism , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/chemistry , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Line , Cysteine/genetics , Electrophysiology , Humans , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/genetics , Mice , Molecular Sequence Data , Oxidation-Reduction , Patch-Clamp Techniques , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.
Subject(s)
Alternative Splicing , Large-Conductance Calcium-Activated Potassium Channels/chemistry , Large-Conductance Calcium-Activated Potassium Channels/physiology , Protein Subunits/metabolism , Amino Acid Sequence , Animals , Biophysical Phenomena , Biophysics , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Endoplasmic Reticulum/metabolism , Evolution, Molecular , Exons , Fluorescent Antibody Technique, Indirect , Humans , Immunohistochemistry , Introns , Large-Conductance Calcium-Activated Potassium Channels/genetics , Mice , Microscopy, Confocal , Molecular Sequence Data , Patch-Clamp Techniques , Phosphorylation , Precipitin Tests , Protein Subunits/chemistry , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. We validated this approach by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Finally, we present strong evidence showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo.
