The pore-forming ␣-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 ⌬e23. 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. ⌬e23 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 ␣-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 M [Ca 2؉ ] 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 ␣-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.Large conductance calcium-and voltage-activated potassium (BK) 3 channels are uniquely regulated by changes in both transmembrane potential as well as intracellular free calcium levels (1). They are widely expressed and thus play an important role in the modulation of cellular excitability in many tissues. Hence, they control diverse physiological processes, including regulation of vascular tone (2-4), micturition (5), neuronal excitability (6, 7), neurotransmitter release (8, 9), endocrine function (10 -12), innate immunity (13), and hearing (14, 15).BK channels in native tissues display a physiologically diverse array of phenotypes. Even neighboring cells (16, 17), or compartments within cells (18,19), may express BK channels with differences in their functional properties. Furthermore, these properties can be modified temporally, for example, during development (20 -22) or following a physiological challenge (23-27).At least two major post-transcriptional mechanisms are involved in generating such functional diversity as follows: alternative pre-mRNA splicing of BK channel pore-forming ␣-subunits and assembly of ␣-subunits with a family of transmembrane modulatory ␤-subunits. Although ␣-subunits are encoded by a single gene (1, 28 -30) (KCNMA1, also referred to as Slo), ␤-subunits are encoded by four distinct genes (KCNMB1-4) (31-34).Several sites o...

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 Caactivated 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 ␣-subunits provides a powerful mechanism for generating functional diversity. Here, we demonstrate that the hypoxia sensitivity of BK channel ␣-subunits is splicevariant-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.alternative splicing ͉ KCNMA1 ͉ oxygen sensing M ammalian cell survival depends on the presence of oxygen. The lowering of oxygen tension (hypoxia) (whether from the disruption of blood flow, inhibition of gaseous exchange, or changes in cellular metabolism) can trigger a range of physiological responses that attempt to minimize the detrimental effects of hypoxia. Large-conductance Ca-and voltage-activated K (BK) channels have been identified as one of the key mediators of the response of the body to hypoxia. BK channels are important for the ''oxygen-sensing'' function of specialized tissues, such as the carotid body and neuroepithelia (1-3), as well as for determining cellular excitability in smooth muscle and neurons (4, 5). However, the responsiveness of native BK channels to changes in oxygen tension is as diverse as the tissues in which they are expressed, with some being completely insensitive to hypoxia (6) and others being potently inhibited by hypoxia (1-3). Also, cellular and tissue sensitivity to hypoxia are highly plastic (7-9), with adaptive responses that depend on prior and prevailing conditions, which may involve changes in BK channel expression (10), although the underlying mechanisms are essentially unknown.The pore-forming ␣-subunits of BK channels are encoded by a single gene (11), KCNMA1, which undergoes extensive alternative pre-mRNA splicing (12, 13). The ␣-subunits assemble as tetramers to form functional channels (14, 15). Distinct splicevariant mRNAs of ␣-subunits may be expressed in the same cell or differentially expressed between tissues or even neighboring cells (16,17). Dynamic modification of splice-variant mRNA expression (18, 19) allows plasticity in BK channel phenotype and cellular regulation (20)(21)(22). Functional ...

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.