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...

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 ...

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discslarge/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.phospholemman | sodium pump | palmitoylation | DHHC | ion transport P rotein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol via a thioester bond, is catalyzed by Asp-His-His-Cys motif-containing palmitoyl acyltransferases (DHHC-PATs); there are 23 human isoforms (1). These zinc-finger-containing enzymes typically have four transmembrane (TM) domains, with a conserved ∼50 amino acid cysteine-rich cytosolic core located between TM2 and -3, which contains a conserved DHHC motif, the active site. In contrast, the intracellular amino and carboxyl termini are poorly conserved, and likely contribute to DHHC isoform substrate selectivity (1). DHHC-PATs are expressed throughout the secretory pathway, but DHHC5 is widely recognized as one of very few cell-surfacelocalized PATs (2, 3). The final four amino acids of DHHC5 form a canonical class II PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, which interacts with postsynaptic density protein 95 (PSD-95) (2), although PSD-95 is not itself a DHHC5 substrate.An appreciation is now growing that protein palmitoylation turns over rapidly (in minutes) for certain proteins (4-8). For example, dynamic surface membrane protein palmitoylation by DHHC5 underlies a novel form of endocytosis, massive endocytosis (MEND), in which up to 70% of the cell surface membrane is internalized (7, 8). Calci...

Background: Enzymes controlling (de)palmitoylation of ion channels are poorly defined.Results: Palmitoylation of BK channels by zDHHC22 and zDHHC23 and depalmitoylation by LYPLA1 and LYPLAL1 controls BK channel cell surface expression.Conclusion: Acyl protein transferases and thioesterases display substrate specificity and control BK channel surface expression.Significance: Understanding how channels are (de)palmitoylated is essential for defining the role of palmitoylation in ion channel physiology.

Large conductance voltage-and calcium-activated potassium (BKCa) channels are important signaling molecules that are regulated by multiple protein kinases and protein phosphatases at multiple sites. The pore-forming ␣-subunits, derived from a single gene that undergoes extensive alternative pre-mRNA splicing, assemble as tetramers. Although consensus phosphorylation sites have been identified within the C-terminal domain of ␣-subunits, it is not known whether phosphorylation of all or single ␣-subunits within the tetramer is required for functional regulation of the channel. Here, we have exploited a strategy to study single-ion channels in which both the ␣-subunit splice-variant composition is defined and the number of consensus phosphorylation sites available within each tetramer is known. We have used this approach to demonstrate that cAMP-dependent protein kinase (PKA) phosphorylation of the conserved C-terminal PKA consensus site (S899) in all four ␣-subunits is required for channel activation. In contrast, inhibition of BK Ca channel activity requires phosphorylation of only a single ␣-subunit at a splice insert (STREX)-specific PKA consensus site (S4 STREX). Thus, distinct modes of BKCa channel regulation by PKA phosphorylation exist: an ''all-or-nothing'' rule for activation and a ''single-subunit'' rule for inhibition. This essentially digital regulation has important implications for the combinatorial and conditional regulation of BK Ca channels by reversible protein phosphorylation.L arge conductance voltage-and calcium-activated potassium (BK Ca ) channels are important regulators of cellular function in the endocrine, nervous, cardiovascular, and immune systems (1-6). BK Ca channels are assembled as tetramers (7, 8) of pore-forming ␣-subunits encoded by a single gene (9) that undergoes extensive alternative splicing (10, 11). Distinct ␣-subunit splice-variant mRNAs may be expressed in the same cell, differentially expressed between tissues, or even neighboring cells (12, 13), and dynamic modification of splice-variant mRNA expression (14, 15) may result in altered BK Ca channel phenotype and cellular regulation (5).Similar to other tetrameric potassium channels BK Ca channels are potently regulated by a variety of serine͞threonine protein kinases (16), including cAMP-dependent protein kinase (PKA) (10,(17)(18)(19)(20). The functional response of BK Ca channels to PKA phosphorylation depends on the splice-variant ␣-subunit composition of the tetramer (10, 19). For example, PKA activates homotetramers of mammalian ZERO splice variants (10,17,19), whereas PKA inhibits homotetramers of STREX variants (10). This differential regulation of BK Ca channels by PKA depends on functional consensus of PKA phosphorylation sites within the C terminus of the ␣-subunit. PKA activation of ZERO variants requires a functional conserved C-terminal PKA site (S899) (10,17,19), whereas PKA inhibition of STREX requires a functional PKA site (S4 STREX ) within the STREX insert (10).However, it is unknown how many ␣-subunits...