Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase.

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

Palmitoylation of the S0-S1 linker regulates cell surface expression of voltage- and calcium-activated potassium (BK) channels.

S-palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Membrane trafficking of large conductance calcium-activated potassium channels is regulated by alternative splicing of a transplantable, acidic trafficking motif in the RCK1-RCK2 linker.

Trafficking of the pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSloDelta(579-664), in which these trafficking signals are excluded because of in-frame exon skipping. The hSloDelta(579-664) variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and downstream trafficking signals. Functionally, the hSloDelta(579-664) variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

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 pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-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.

CD31 delays phagocyte membrane repolarization to promote efficient binding of apoptotic cells.

Homophilic ligation of CD31, a member of the Ig superfamily of adhesion receptors, promotes macrophage clearance of apoptotic leukocytes by a mechanism hitherto not described. In studying CD31-dependent regulation of beta1-integrin binding of fibronectin-coated Latex beads, we discovered a role for the voltage-gated potassium channel ether-à-go-go-related gene (ERG) as a downstream effector of CD31 signaling. ERG was identified by tandem mass spectrometry as a 140-kDa protein, which was selectively modified with biotin following the targeted delivery of a biotin-transfer reagent to CD31 using Fab fragments of an anti-CD31 mAb. Similar results were obtained with macrophages but not K562 cells, expressing a truncated cytoplasmic tail of CD31, which failed to regulate bead binding. Colocalization of CD31 with ERG was confirmed by immunofluorescence for K562 cells and macrophages. We now demonstrate that the resting membrane potential of macrophages is depolarized on contact with apoptotic cells and that CD31 inhibits the ERG current, which would otherwise function to repolarize. Sustained depolarization favored the firm binding of phagocytic targets, a prerequisite for efficient engulfment. Our results identify ERG as a downstream effector of CD31 in the regulation of integrin-dependent binding of apoptotic cells by macrophages.

A cysteine-rich motif confers hypoxia sensitivity to mammalian large conductance voltage- and Ca-activated K (BK) channel alpha-subunits.

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.

Conditional protein phosphorylation regulates BK channel activity in rat cerebellar Purkinje neurons.

Large conductance calcium- and voltage-activated potassium (BK) channels are widely expressed in the mammalian central nervous system. Although the activity of BK channels in endocrine and vascular cells is regulated by protein kinases and phosphatases associated with the channel complex, direct evidence for such modulation in neurons is largely lacking. Single-channel analysis from inside-out patches isolated from the soma of dissociated rat cerebellar Purkinje neurons demonstrated that the activity of BK channels is regulated by multiple endogenous protein kinases and protein phosphatases in the membrane patch. The majority of BK channels were non-inactivating and displayed a 'low' activity phenotype determined at +40 mV and 1 muM intracellular free calcium. These channels were activated by cAMP-dependent protein kinase (PKA) associated with the patch and the extent of PKA activation was limited by an opposing endogenous type 2A-like protein phosphatase (PP2A). Importantly, PKA activation was dependent upon the prior phosphorylation status of the BK channel complex dynamically controlled by protein kinase C (PKC) and protein phosphatase 1 (PP1). In contrast, Purkinje cells also displayed a low proportion of non-inactivating BK channels with a 'high' activity under the same recording conditions and these channels were inhibited by endogenous PKA. Our data suggest that: (1) multiple endogenous protein kinases and phosphatases functionally couple to the BK channel complex to allow conditional modulation of BK channel activity in neurons, and (2) native, phenotypically distinct, neuronal BK channels are differentially sensitive to PKA-dependent phosphorylation.