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...

Large-conductance calcium- and voltage- activated potassium (BK) channels play a fundamental role in the signaling pathways regulating mouse anterior pituitary corticotrope function. Here we describe the cloning and functional characterization of the components of mouse corticotrope BK channels. RT-PCR cloning and splice variant analysis of mouse AtT20 D16:16 corticotropes revealed robust expression of mslo transcripts encoding pore-forming alpha-subunits containing the mouse homolog of the 59-amino acid STREX-1 exon at splice site 2. RT-PCR and functional analysis, using the triterpenoid glycoside, DHS-1, revealed that native corticotrope BK channels are not functionally coupled to beta-subunits in vivo. Functional expression of the STREX-1 containing alpha-subunit in HEK 293 cells resulted in BK channels with calcium sensitivity, single-channel conductance, and inhibition by protein kinase A identical to that of native mouse corticotrope BK channels. This report represents the first corticotrope ion channel to be characterized at the molecular level and demonstrates that mouse corticotrope BK channels are composed of alpha-subunits expressing the mouse STREX-1 exon.

Many intracellular membranes contain ion channels, although their physiological roles are often poorly understood. In this study we incorporated single anion channels colocalized with rat brain endoplasmic reticulum (ER) ryanodine-sensitive Ca(2+)-release channels into planar lipid bilayers. The channels opened in bursts, with more activity at negative (cytoplasm-ER lumen) membrane potentials, and they occupied four open conductance levels with frequencies well described by the binomial equation. The probability of a protomer being open decreased from approximately 0.7 at -40 mV to approximately 0.2 at +40 mV, and the channels selected between different anions in the order PSCN > PNO3 > PBr > PCl > PF. They were also permeant to cations, including the large cation Tris+ (PTris/PCl = 0.16). Their conductance saturated at 170 pS in choline Cl. The channels were inactivated by 15 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and blocked with low affinity (KD of 1-100 microM) by anthracene-9-carboxylic acid, ethacrynic acid, frusemide (furosemide), HEPES, the indanyloxyacetic acid derivative IAA-94, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and Zn2+. Unlike protein translocation pores, the channels were unaffected by high salt concentrations or puromycin. They may regulate ER Ca2+ release, or be channel components en route to their final cellular destinations. Alternatively, they may contribute to the fusion machinery involved in intracellular membrane trafficking.

Large conductance calcium-and voltage-activated potassium (BK) channels play a fundamental role in many physiological processes including action potential repolarization, modulation of calcium signals, hormone release and regulation of vascular tone (