Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.Large conductance calcium-and voltage-activated potassium (BK) 1 channels link intracellular chemical signaling events with the electrical properties of excitable cells in the endocrine, nervous, and vascular systems (1-3). BK channels are further potently modulated by reversible protein phosphorylation (4 -7). In native tissues BK channels display considerable diversity and plasticity in their regulation by reversible protein phosphorylation. For example, cAMP-dependent protein kinase (PKA) phosphorylation activates BK channels in smooth muscle cells and many neurones but inhibits channel activity in endocrine cells of the anterior pituitary (5, 7-11). Furthermore, the direction of channel regulation by PKA can be modified during challenges to homeostasis (9 -11).The pore-forming ␣-subunits of BK channels are derived from a single gene (Slo) that undergoes extensive alternative splicing to produce channels with distinct phenotypes (12-15). Importantly, alternative splicing of the ␣-subunit is dynamically regulated in adults, for example during stress or pregnancy (15, 16). Thus the diversity and plasticity of responses to PKA-dependent protein phosphorylation observed between BK channels in native tissues may result either from differential modulation of alternatively spliced BK channel ␣-subunits (12-15) or through their interaction with different signaling complexes and ␤-subunits (17-19).To address whether BK channel alternative splice variants are differentially regulated by PKA-mediated protein phosphorylation, we have examined the regulation of three mouse (mslo) BK channel variants (20 -22) expressed in HEK293 cells. BK channels are regulated by multiple protein kinase signaling pathways (5,19,23,24). We have thus assayed the functional regulation of BK channel splice variants by directly activating PKA that remains closely associated with the channels in excised inside-out patches. EXPERIMENTAL PROCEDURESMolecular and Cell Biology-cDNAs encod...

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...

Neocortical circuitry can alter throughout life with experience. However, the contributions of changes in synaptic strength and modifications in neuronal wiring to experience-dependent plasticity in mature animals remain unclear. We trimmed whiskers of rats and made electrophysiological recordings after whisker cortical maps have developed. Measurements of miniature EPSPs suggested that synaptic inputs to layer 2/3 pyramidal neurons were altered at the junction of deprived and spared cortex in primary somatosensory cortex. Whole-cell recordings were made from pairs of synaptically connected pyramidal neurons to investigate possible changes in local excitatory connections between layer 2/3 pyramidal neurons. The neurons were filled with fluorescent dyes during recording and reconstructed in three dimensions using confocal microscopy and image deconvolution to identify putative synapses. We show that sensory deprivation induces a striking reduction in connectivity between layer 2/3 pyramidal neurons in deprived cortex without large-scale, compensatory increases in the strength of remaining local excitatory connections. A markedly different situation occurs in spared cortex. Connection strength is potentiated, but local excitatory connectivity and synapse number per connection are unchanged. Our data suggest that alterations in local excitatory circuitry enhance the expansion of spared representations into deprived cortex. Moreover, our findings offer one explanation for how the responses of spared and deprived cortex to sensory deprivation can be dissociated in developed animals.

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of thenervoussystemandN-methyl-D-aspartate(NMDA)receptordependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by ≈30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 M glutamate but not by 30 M glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.