The human Kv1.1 channel is palmitoylated, modulating voltage sensing: Identification of a palmitoylation consensus sequence

Gubitosi-Klug, Rose; Mancuso, David J.; Gross, Richard W.

doi:10.1073/pnas.0501999102

Cited by 65 publications

(66 citation statements)

References 47 publications

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“…This raises the possibility that regulation of delayed rectifier, voltage-gated K channels by iPLA 2 ␤, or products of its action might be a phenomenon that is not confined to ␤-cells. Kv1.1 channel activity (66) and subcellular location (67) are also affected by free fatty acids, and it is possible that colocalization or direct interaction of iPLA 2 ␤ and voltage-gated K channels, such as Kv1.1 and Kv2.1, occur.…”

Section: Discussionmentioning

confidence: 99%

Modulation of the Pancreatic Islet β-Cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

Jacobson

Weber

Bao

et al. 2007

Journal of Biological Chemistry

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Glucose stimulates both insulin secretion and hydrolysis of arachidonic acid (AA) esterified in membrane phospholipids of pancreatic islet ␤-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet ␤-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 M) and subjected to graded degrees of depolarization exhibit a significantly shorter time-to-peak current interval than do control cells. AA causes a rapid decay and reduced peak conductance of delayed rectifier currents from INS-1 cells and from primary ␤-cells isolated from mouse, rat, and human pancreatic islets. Stimulating mouse islets with AA results in a significant increase in the frequency of glucoseinduced [Ca 2؉ ] oscillations, which is an expected effect of Kv2.1 channel blockade. Stimulation with concentrations of glucose and carbachol that accelerate hydrolysis of endogenous AA from islet phosphoplipids also results in accelerated Kv2.1 inactivation and a shorter time-to-peak current interval. Group VIA phospholipase A 2 (iPLA 2 ␤) hydrolyzes ␤-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA 2 ␤ prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in ␤-cells from iPLA 2 ␤ ؊/؊ mice. Stably transfected INS-1 cells that overexpress iPLA 2 ␤ hydrolyze phospholipids more rapidly than control INS-1 cells and also exhibit an increase in the inactivation rate of the delayed rectifier currents. These results suggest that Kv2.1 currents could be dynamically modulated in the pancreatic islet ␤-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca 2؉ entry and insulin secretion.Glucose metabolism within the pancreatic islet ␤-cell generates a multitude of signals that regulate insulin secretion. Changes in the relative concentrations of the metabolites ATP and ADP cause ␤-cell depolarization via closure of ATP-sensitive potassium channels (K ATP ), 3 which results in activation of voltage-operated Ca 2ϩ channels, Ca 2ϩ influx, and insulin secretion (1). The extent of ␤-cell depolarization and insulin release are regulated in part by the activation of repolarizing ion channels, including the voltage-gated potassium channel, Kv2.1 (2-4). One mechanism employed by pancreatic ␤-cells to regulate the biophysical activity of the ion channels involved in insulin release involves hydrolysis of membrane phospholipids to yield mediators that include inositol triphosphates and free fatty acids (5-8).Pharmacologic, biochemical, and genetic evidence suggests that glucose-stimulated hydrolysis of esterified arachidonic acid from ␤-cell membrane phospholipids is required for physiological insulin secretion (7-25). Pancreatic islet ␤-cells contain high levels of arachidonic ac...

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Section: Discussionmentioning

confidence: 99%

Modulation of the Pancreatic Islet β-Cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

Jacobson

Weber

Bao

et al. 2007

Journal of Biological Chemistry

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“…Protein phosphorylation is a fundamental mechanism to control ion channel function, and increasing evidence suggests that multiple ion channels are also regulated by palmitoylation (16)(17)(18)(19)(20)(21). We would predict that phosphorylationpalmitoylation cross-talk is likely to occur when sites of palmitoylation are flanked by sites of serine/threonine phosphorylation.…”

Section: Pka Phosphorylation Dissociates the Strex C Terminus From Thementioning

confidence: 99%

“…Catalytic subunit of PKAc was from Promega. The 8-CPT-cGMP, 8-CPT-cAMP, KT5823, chelerythrine chloride, KN-62, and PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] were from Calbiochem.…”

Section: Palmitoylation Prediction and Palmitoylation Assays Css-palmentioning

confidence: 99%

“…1A). In many proteins, including other voltage-and ligand-gated ion channels (16)(17)(18)(19)(20)(21), cysteine residues are common targets for protein palmitoylation, the covalent attachment of a palmitate lipid to a cysteine residue via a thioester bond. Palmitoylation can exert diverse effects on protein function including allowing cytosolic protein domains to anchor to the plasma membrane (22)(23)(24).…”

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confidence: 99%

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Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Tian

Jeffries

McClafferty

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

105

180

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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 ...

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“…Although ion channels are integral parts of the cell signaling machinery, only a few studies have identified ion channel palmitoylation (13,(19)(20)(21)(22). Furthermore, no information on direct myristoylation of any ion channel has been reported yet.…”

Section: Discussionmentioning

confidence: 99%

Unconventional myristoylation of large-conductance Ca ²⁺ -activated K ⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression

Alioua

Li²,

Wu³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Protein myristoylation is a means by which cells anchor proteins into membranes. The most common type of myristoylation occurs at an N-terminal glycine. However, myristoylation rarely occurs at an internal amino acid residue. Here we tested whether the α-subunit of the human large-conductance voltage-and Ca 2+ -activated K + channel (hSlo1) might undergo internal myristoylation. hSlo1 expressed in HEK293T cells incorporated [ 3 H]myristic acid via a posttranslational mechanism, which is insensitive to cycloheximide, an inhibitor of protein biosynthesis. In-gel hydrolysis of [ 3 H]myristoyl-hSlo1 with alkaline NH 2 OH (which cleaves hydroxyesters) but not neutral NH 2 OH (which cleaves thioesters) completely removed [ 3 H]myristate from hSlo1, suggesting the involvement of a hydroxyester bond between hSlo1's hydroxyl-bearing serine, threonine, and/or tyrosine residues and myristic acid; this type of esterification was further confirmed by its resistance to alkaline Tris·HCl. Treatment of cells expressing hSlo1 with 100 μM myristic acid caused alteration of hSlo1 activation kinetics and a 40% decrease in hSlo1 current density from 20 to 12 nA*MΩ. Immunocytochemistry confirmed a decrease in hSlo1 plasmalemma localization by myristic acid. Replacement of the six serines or the seven threonines (but not of the single tyrosine) of hSlo1 intracellular loops 1 and 3 with alanines decreased hSlo1 direct myristoylation by 40-44%, whereas in combination decreased myristoylation by nearly 90% and abolished the myristic acid-induced change in current density. Our data demonstrate that an ion channel, hSlo1, is internally and posttranslationally myristoylated. Myristoylation occurs mainly at hSlo1 intracellular loop 1 or 3, and is an additional mechanism for channel surface expression regulation.MaxiK channel | BK Ca channel | posttranslational modification | traffic T he large-conductance voltage-and calcium-activated potassium channel (MaxiK, BK Ca ) is ubiquitously expressed regulating numerous physiological functions such as neuronal action potential firing, neurotransmitter release, and vascular smooth muscle tone (1). Four pore-forming α-subunits (Slo1, an ∼125-kDa protein) make a functional MaxiK channel. Based on structure/function studies, Slo1 protein can be separated into two main functional cassettes: the multipass transmembranous N terminus (∼40 kDa) and a large cytoplasmic C terminus (∼85 kDa). The N terminus has seven (S0-S6) transmembrane segments, together forming the voltage-sensing and the pore-conducting domains. The intracellular C terminus carries two RCK domains (RCK1, RCK2) and contains the Ca 2+

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The human Kv1.1 channel is palmitoylated, modulating voltage sensing: Identification of a palmitoylation consensus sequence

Cited by 65 publications

References 47 publications

Modulation of the Pancreatic Islet β-Cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

Modulation of the Pancreatic Islet β-Cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Unconventional myristoylation of large-conductance Ca ²⁺ -activated K ⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression

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The human Kv1.1 channel is palmitoylated, modulating voltage sensing: Identification of a palmitoylation consensus sequence

Cited by 65 publications

References 47 publications

Modulation of the Pancreatic Islet β-Cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

Modulation of the Pancreatic Islet β-Cell-delayed Rectifier Potassium Channel Kv2.1 by the Polyunsaturated Fatty Acid Arachidonate

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Unconventional myristoylation of large-conductance Ca 2+ -activated K + channel (Slo1) via serine/threonine residues regulates channel surface expression

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Unconventional myristoylation of large-conductance Ca ²⁺ -activated K ⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression