Publications by authors named "Michael J Shipston"

54 Publications

Regulatory effects of protein S-acylation on insulin secretion and insulin action.

Open Biol 2021 Mar 31;11(3):210017. Epub 2021 Mar 31.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK.

Post-translational modifications (PTMs) such as phosphorylation and ubiquitination are well-studied events with a recognized importance in all aspects of cellular function. By contrast, protein S-acylation, although a widespread PTM with important functions in most physiological systems, has received far less attention. Perturbations in S-acylation are linked to various disorders, including intellectual disability, cancer and diabetes, suggesting that this less-studied modification is likely to be of considerable biological importance. As an exemplar, in this review, we focus on the newly emerging links between S-acylation and the hormone insulin. Specifically, we examine how S-acylation regulates key components of the insulin secretion and insulin response pathways. The proteins discussed highlight the diverse array of proteins that are modified by S-acylation, including channels, transporters, receptors and trafficking proteins and also illustrate the diverse effects that S-acylation has on these proteins, from membrane binding and micro-localization to regulation of protein sorting and protein interactions.
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http://dx.doi.org/10.1098/rsob.210017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061761PMC
March 2021

Site-specific deacylation by ABHD17a controls BK channel splice variant activity.

J Biol Chem 2020 12 10;295(49):16487-16496. Epub 2020 Sep 10.

Centre for Discovery Brain Sciences, Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom. Electronic address:

-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel -acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the -acylated S0-S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct -acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.
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http://dx.doi.org/10.1074/jbc.RA120.015349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864050PMC
December 2020

LINGO1 is a regulatory subunit of large conductance, Ca-activated potassium channels.

Proc Natl Acad Sci U S A 2020 01 13;117(4):2194-2200. Epub 2020 Jan 13.

Smooth Muscle Research Centre, Dundalk Institute of Technology, Louth A81 K584, Ireland;

LINGO1 is a transmembrane protein that is up-regulated in the cerebellum of patients with Parkinson's disease (PD) and Essential Tremor (ET). Patients with additional copies of the LINGO1 gene also present with tremor. Pharmacological or genetic ablation of large conductance Ca-activated K (BK) channels also result in tremor and motor disorders. We hypothesized that LINGO1 is a regulatory BK channel subunit. We show that 1) LINGO1 coimmunoprecipitated with BK channels in human brain, 2) coexpression of LINGO1 and BK channels resulted in rapidly inactivating BK currents, and 3) LINGO1 reduced the membrane surface expression of BK channels. These results suggest that LINGO1 is a regulator of BK channels, which causes a "functional knockdown" of these currents and may contribute to the tremor associated with increased LINGO1 levels.
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http://dx.doi.org/10.1073/pnas.1916715117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994976PMC
January 2020

-Acylation controls functional coupling of BK channel pore-forming α-subunits and β1-subunits.

J Biol Chem 2019 08 18;294(32):12066-12076. Epub 2019 Jun 18.

Centre for Discovery Brain Sciences, Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom. Electronic address:

The properties and physiological function of pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are potently modified by their functional coupling with regulatory subunits in many tissues. However, mechanisms that might control functional coupling are very poorly understood. Here we show that -acylation, a dynamic post-translational lipid modification of proteins, of the intracellular S0-S1 loop of the BK channel pore-forming α-subunit controls functional coupling to regulatory β1-subunits. In HEK293 cells, α-subunits that cannot be -acylated show attenuated cell surface expression, but expression was restored by co-expression with the β1-subunit. However, we also found that nonacylation of the S0-S1 loop reduces functional coupling between α- and β1-subunits by attenuating the β1-subunit-induced left shift in the voltage for half-maximal activation. In mouse vascular smooth muscle cells expressing both α- and β1-subunits, BK channel α-subunits were endogenously -acylated. We further noted that -acylation is significantly reduced in mice with a genetic deletion of the palmitoyl acyltransferase (Zdhhc23) that controls -acylation of the S0-S1 loop. Genetic deletion of Zdhhc23 or broad-spectrum pharmacological inhibition of -acylation attenuated endogenous BK channel currents independently of changes in cell surface expression of the α-subunit. We conclude that functional effects of -acylation on BK channels depend on the presence of β1-subunits. In the absence of β1-subunits, -acylation promotes cell surface expression, whereas in its presence, -acylation controls functional coupling. -Acylation thus provides a mechanism that dynamically regulates the functional coupling with β1-subunits, enabling an additional level of conditional, cell-specific control of ion-channel physiology.
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http://dx.doi.org/10.1074/jbc.RA119.009065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690687PMC
August 2019

siRNA Knockdown of Mammalian zDHHCs and Validation of mRNA Expression by RT-qPCR.

Methods Mol Biol 2019 ;2009:151-168

Centre for Discovery Brain Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.

The lack of specific pharmacological tools to interrogate the functional role of palmitoyl acyltransferases (zDHHCs) in mammalian cells has significantly hampered the understanding of this important gene family. Gene silencing by RNA interference (RNAi) is a process in eukaryotes that allows specific knockdown of the expression of proteins by targeting their coding mRNA. RNAi can thus be used as a proteomic tool to study the functional role of specific zDHHCs in cells by analyzing the effects of endogenous zDHHC knockdown on their protein targets or pathways. Here we describe the application of short interfering RNA (siRNA), a class of short (20-25 base pairs) double-stranded RNAs, to knockdown endogenous zDHHC enzymes expressed in human embryonic kidney (HEK293) cells and subsequent validation of knockdown efficiency using RT-qPCR to quantify zDHHC mRNA levels.
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http://dx.doi.org/10.1007/978-1-4939-9532-5_12DOI Listing
March 2020

Control of anterior pituitary cell excitability by calcium-activated potassium channels.

Mol Cell Endocrinol 2018 03 5;463:37-48. Epub 2017 Jun 5.

Centre for Integrative Physiology, Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, Edinburgh, EH89XD, UK. Electronic address:

In anterior pituitary endocrine cells, large (BK), small (SK) and intermediate (IK) conductance calcium activated potassium channels are key determinants in shaping cellular excitability in a cell type- and context-specific manner. Indeed, these channels are targeted by multiple signaling pathways that stimulate or inhibit cellular excitability. BK channels can, paradoxically, both promote electrical bursting as well as terminate bursting and spiking dependent upon intrinsic BK channel properties and proximity to voltage gated calcium channels in somatotrophs, lactotrophs and corticotrophs. In contrast, SK channels are predominantly activated by calcium released from intracellular IP3-sensitive calcium stores and mediate membrane hyperpolarization in cells including gonadotrophs and corticotrophs. IK channels are predominantly expressed in corticotrophs where they limit membrane excitability. A major challenge for the future is to determine the cell-type specific molecular composition of calcium-activated potassium channels and how they control anterior pituitary hormone secretion as well as other calcium-dependent processes.
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http://dx.doi.org/10.1016/j.mce.2017.06.003DOI Listing
March 2018

Distinct domains of the β1-subunit cytosolic N terminus control surface expression and functional properties of large-conductance calcium-activated potassium (BK) channels.

J Biol Chem 2017 05 3;292(21):8694-8704. Epub 2017 Apr 3.

From the Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom,

The properties and function of large-conductance calcium- and voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory β1-subunits. Although the short cytosolic N-terminal domain of the β1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N terminus control these distinct processes remains unknown. Here we demonstrate that the first six N-terminal residues including Lys-3, Lys-4, and Leu-5 are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane-distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane-spanning domain (TM1) of the β1-subunit. In contrast, a membrane-proximal leucine residue (Leu-17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient endoplasmic reticulum exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the β1-subunit N terminus.
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http://dx.doi.org/10.1074/jbc.M116.769505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448097PMC
May 2017

Heterogeneity of Calcium Responses to Secretagogues in Corticotrophs From Male Rats.

Endocrinology 2017 06;158(6):1849-1858

Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.

Heterogeneity in homotypic cellular responses is an important feature of many biological systems, and it has been shown to be prominent in most anterior pituitary hormonal cell types. In this study, we analyze heterogeneity in the responses to hypothalamic secretagogues in the corticotroph cell population of adult male rats. Using the genetically encoded calcium indicator GCaMP6s, we determined the intracellular calcium responses of these cells to corticotropin-releasing hormone and arginine-vasopressin. Our experiments revealed marked population heterogeneity in the response to these peptides, in terms of amplitude and dynamics of the responses, as well as the sensitivity to different concentrations and duration of stimuli. However, repeated stimuli to the same cell produced remarkably consistent responses, indicating that these are deterministic on a cell-by-cell level. We also describe similar heterogeneity in the sensitivity of cells to inhibition by corticosterone. In summary, our results highlight a large degree of heterogeneity in the cellular mechanisms that govern corticotroph responses to their physiological stimuli; this could provide a mechanism to extend the dynamic range of the responses at the population level to allow adaptation to different physiological challenges.
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http://dx.doi.org/10.1210/en.2017-00107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460926PMC
June 2017

Obesogenic and Diabetogenic Effects of High-Calorie Nutrition Require Adipocyte BK Channels.

Diabetes 2016 Dec 7;65(12):3621-3635. Epub 2016 Sep 7.

Pharmakologie, Toxikologie und Klinische Pharmazie, Institut für Pharmazie, Tübingen, Germany

Elevated adipose tissue expression of the Ca- and voltage-activated K (BK) channel was identified in morbidly obese men carrying a BK gene variant, supporting the hypothesis that K channels affect the metabolic responses of fat cells to nutrients. To establish the role of endogenous BKs in fat cell maturation, storage of excess dietary fat, and body weight (BW) gain, we studied a gene-targeted mouse model with global ablation of the BK channel (BK) and adipocyte-specific BK-deficient (adipoqBK) mice. Global BK deficiency afforded protection from BW gain and excessive fat accumulation induced by a high-fat diet (HFD). Expansion of white adipose tissue-derived epididymal BK preadipocytes and their differentiation to lipid-filled mature adipocytes in vitro, however, were improved. Moreover, BW gain and total fat masses of usually superobese ob/ob mice were significantly attenuated in the absence of BK, together supporting a central or peripheral role for BKs in the regulatory system that controls adipose tissue and weight. Accordingly, HFD-fed adipoqBK mutant mice presented with a reduced total BW and overall body fat mass, smaller adipocytes, and reduced leptin levels. Protection from pathological weight gain in the absence of adipocyte BKs was beneficial for glucose handling and related to an increase in body core temperature as a result of higher levels of uncoupling protein 1 and a low abundance of the proinflammatory interleukin-6, a common risk factor for diabetes and metabolic abnormalities. This suggests that adipocyte BK activity is at least partially responsible for excessive BW gain under high-calorie conditions, suggesting that BK channels are promising drug targets for pharmacotherapy of metabolic disorders and obesity.
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http://dx.doi.org/10.2337/db16-0245DOI Listing
December 2016

Glucocorticoids Inhibit CRH/AVP-Evoked Bursting Activity of Male Murine Anterior Pituitary Corticotrophs.

Endocrinology 2016 08 2;157(8):3108-21. Epub 2016 Jun 2.

Centre for Integrative Physiology (P.J.D., M.J.S.), College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; Biomedical Neuroscience Research Group (J.T.), University of Exeter Medical School, Exeter EX4 4PL, United Kingdom; Division of Pharmacology, Toxicology, and Clinical Pharmacy (P.R.), Institute for Pharmacy, University of Tübingen, D-72076 Tübingen, Germany; and Department of Mathematics and Programs in Neuroscience and Molecular Biophysics (R.B.), Florida State University, Tallahassee, Florida 32306.

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which governs the neuroendocrine response to stress. Corticotrophs are electrically excitable and fire spontaneous single-spike action potentials and also display secretagogue-induced bursting behavior. The HPA axis function is dependent on effective negative feedback in which elevated plasma glucocorticoids result in inhibition at the level of both the pituitary and the hypothalamus. In this study, we have used an electrophysiological approach coupled with mathematical modeling to investigate the regulation of spontaneous and CRH/arginine vasopressin-induced activity of corticotrophs by glucocorticoids. We reveal that pretreatment of corticotrophs with 100 nM corticosterone (CORT; 90 and 150 min) reduces spontaneous activity and prevents a transition from spiking to bursting after CRH/arginine vasopressin stimulation. In addition, previous studies have identified a role for large-conductance calcium- and voltage-activated potassium (BK) channels in the generation of secretagogue-induced bursting in corticotrophs. Using the dynamic clamp technique, we demonstrated that CRH-induced bursting can be switched to spiking by subtracting a fast BK current, whereas the addition of a fast BK current can induce bursting in CORT-treated cells. In addition, recordings from BK knockout mice (BK(-/-)) revealed that CORT can also inhibit excitability through BK-independent mechanisms to control spike frequency. Thus, we have established that glucocorticoids can modulate multiple properties of corticotroph electrical excitability through both BK-dependent and BK-independent mechanisms.
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http://dx.doi.org/10.1210/en.2016-1115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967125PMC
August 2016

The physiology of protein S-acylation.

Physiol Rev 2015 Apr;95(2):341-76

Strathclyde Institute of Pharmacy and Biomedical Sciences, Strathclyde University, Glasgow, United Kingdom; and Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, United Kingdom.

Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field.
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http://dx.doi.org/10.1152/physrev.00032.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551212PMC
April 2015

Large conductance Ca²⁺-activated K⁺ (BK) channels promote secretagogue-induced transition from spiking to bursting in murine anterior pituitary corticotrophs.

J Physiol 2015 Mar 23;593(5):1197-211. Epub 2015 Jan 23.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, EH8 9XD, UK.

Anterior pituitary corticotroph cells are a central component of the hypothalamic-pituitary-adrenal (HPA) axis essential for the neuroendocrine response to stress. Corticotrophs are excitable cells that receive input from two hypothalamic secretagogues, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) to control the release of adrenocorticotrophic hormone (ACTH). Although corticotrophs are spontaneously active and increase in excitability in response to CRH and AVP the patterns of electrical excitability and underlying ionic conductances are poorly understood. In this study, we have used electrophysiological, pharmacological and genetic approaches coupled with mathematical modelling to investigate whether CRH and AVP promote distinct patterns of electrical excitability and to interrogate the role of large conductance calcium- and voltage-activated potassium (BK) channels in spontaneous and secretagogue-induced activity. We reveal that BK channels do not play a significant role in the generation of spontaneous activity but are critical for the transition to bursting in response to CRH. In contrast, AVP promotes an increase in single spike frequency, a mechanism independent of BK channels but dependent on background non-selective conductances. Co-stimulation with CRH and AVP results in complex patterns of excitability including increases in both single spike frequency and bursting. The ability of corticotroph excitability to be differentially regulated by hypothalamic secretagogues provides a mechanism for differential control of corticotroph excitability in response to different stressors.
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http://dx.doi.org/10.1113/jphysiol.2015.284471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358680PMC
March 2015

Large conductance Ca -activated K channels (BK) promote secretagogue-induced transition from spiking to bursting in murine anterior pituitary corticotrophs.

J Physiol 2014 Dec 24. Epub 2014 Dec 24.

Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, University of Edinburgh, Edinburgh, EH89XD, UK.

Anterior pituitary corticotroph cells are a central component of the hypothalamic-pituitary-adrenal (HPA) axis essential for the neuroendocrine response to stress. Corticotrophs are excitable cells that receive input from two hypothalamic secretagogues, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) to control the release of adrenocorticotrophin hormone (ACTH). Although corticotrophs are spontaneously active and increase in excitability in response to CRH and AVP the patterns of electrical excitability and underlying ionic conductances are poorly understood. In this study, we have used electrophysiological, pharmacological and genetic approaches coupled with mathematical modeling to investigate whether CRH and AVP promote distinct patterns of electrical excitability and to interrogate the role of large conductance calcium- and voltage-activated (BK) channels in spontaneous and secretagogue-induced activity. We reveal that BK channels do not play a significant role in the generation of spontaneous activity but are critical for the transition to bursting in response to CRH. In contrast, AVP promotes an increase in single spike frequency, a mechanism independent of BK channels but dependent on background non-selective conductances. Co-stimulation with CRH and AVP results in complex patterns of excitability including increases in both single spike frequency and bursting. The ability of corticotroph excitability to be differentially regulated by hypothalamic secretagogues provides a mechanism for differential control of corticotroph excitability in response to different stressors. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1113/jphysiol.2014.284471DOI Listing
December 2014

Substrate recognition by the cell surface palmitoyl transferase DHHC5.

Proc Natl Acad Sci U S A 2014 Dec 24;111(49):17534-9. Epub 2014 Nov 24.

Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom;

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/Discs-large/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.
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http://dx.doi.org/10.1073/pnas.1413627111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267385PMC
December 2014

S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels.

Front Physiol 2014 5;5:281. Epub 2014 Aug 5.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh Edinburgh, UK.

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease.
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http://dx.doi.org/10.3389/fphys.2014.00281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122160PMC
August 2014

Ion channel regulation by protein S-acylation.

J Gen Physiol 2014 Jun 12;143(6):659-78. Epub 2014 May 12.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD Scotland, UK

Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease.
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http://dx.doi.org/10.1085/jgp.201411176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035745PMC
June 2014

Palmitoylation of the β4-subunit regulates surface expression of large conductance calcium-activated potassium channel splice variants.

J Biol Chem 2013 May 16;288(18):13136-44. Epub 2013 Mar 16.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom.

Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels.
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http://dx.doi.org/10.1074/jbc.M113.461830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642354PMC
May 2013

Regulation of large conductance calcium- and voltage-activated potassium (BK) channels by S-palmitoylation.

Biochem Soc Trans 2013 Feb;41(1):67-71

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD, UK.

BK (large conductance calcium- and voltage-activated potassium) channels are important determinants of physiological control in the nervous, endocrine and vascular systems with channel dysfunction associated with major disorders ranging from epilepsy to hypertension and obesity. Thus the mechanisms that control channel surface expression and/or activity are important determinants of their (patho)physiological function. BK channels are S-acylated (palmitoylated) at two distinct sites within the N- and C-terminus of the pore-forming α-subunit. Palmitoylation of the N-terminus controls channel trafficking and surface expression whereas palmitoylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. Recent studies are beginning to reveal mechanistic insights into how palmitoylation controls channel trafficking and cross-talk with phosphorylation-dependent signalling pathways. Intriguingly, each site of palmitoylation is regulated by distinct zDHHCs (palmitoyl acyltransferases) and APTs (acyl thioesterases). This supports that different mechanisms may control substrate specificity by zDHHCs and APTs even within the same target protein. As palmitoylation is dynamically regulated, this fundamental post-translational modification represents an important determinant of BK channel physiology in health and disease.
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http://dx.doi.org/10.1042/BST20120226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4003526PMC
February 2013

Palmitoylation and membrane association of the stress axis regulated insert (STREX) controls BK channel regulation by protein kinase C.

J Biol Chem 2012 Sep 29;287(38):32161-71. Epub 2012 Jul 29.

Division of Experimental Cardiology, Mannheim Medical Faculty, Heidelberg University, D-68167 Mannheim, Germany.

Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser(700), located between the two regulatory domains of K(+) conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ∼50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability.
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http://dx.doi.org/10.1074/jbc.M112.386359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442546PMC
September 2012

Distinct acyl protein transferases and thioesterases control surface expression of calcium-activated potassium channels.

J Biol Chem 2012 Apr 7;287(18):14718-25. Epub 2012 Mar 7.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, EH8 9XD, Scotland.

Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0-S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ∼30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.
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http://dx.doi.org/10.1074/jbc.M111.335547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340283PMC
April 2012

An electrostatic switch controls palmitoylation of the large conductance voltage- and calcium-activated potassium (BK) channel.

J Biol Chem 2012 Jan 14;287(2):1468-77. Epub 2011 Nov 14.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.

Protein palmitoylation is a major dynamic posttranslational regulator of protein function. However, mechanisms that control palmitoylation are poorly understood. In many proteins, palmitoylation occurs at cysteine residues juxtaposed to membrane-anchoring domains such as transmembrane helices, sites of irreversible lipid modification, or hydrophobic and/or polybasic domains. In particular, polybasic domains represent an attractive mechanism to dynamically control protein palmitoylation, as the function of these domains can be dramatically influenced by protein phosphorylation. Here we demonstrate that a polybasic domain immediately upstream of palmitoylated cysteine residues within an alternatively spliced insert in the C terminus of the large conductance calcium- and voltage-activated potassium channel is an important determinant of channel palmitoylation and function. Mutation of basic amino acids to acidic residues within the polybasic domain results in inhibition of channel palmitoylation and a significant right-shift in channel half maximal voltage for activation. Importantly, protein kinase A-dependent phosphorylation of a single serine residue within the core of the polybasic domain, which results in channel inhibition, also reduces channel palmitoylation. These data demonstrate the key role of the polybasic domain in controlling stress-regulated exon palmitoylation and suggests that phosphorylation controls the domain by acting as an electrostatic switch.
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http://dx.doi.org/10.1074/jbc.M111.224840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256903PMC
January 2012

Control of hypothalamic-pituitary-adrenal stress axis activity by the intermediate conductance calcium-activated potassium channel, SK4.

J Physiol 2011 Dec 31;589(Pt 24):5965-86. Epub 2011 Oct 31.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH89XD, Scotland, UK.

The anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4(-/-)). In addition, Kcnn4(-/-) mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4(-/-) mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function.
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http://dx.doi.org/10.1113/jphysiol.2011.219378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286679PMC
December 2011

Ion channel regulation by protein palmitoylation.

J Biol Chem 2011 Mar 7;286(11):8709-16. Epub 2011 Jan 7.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom.

Protein S-palmitoylation, the reversible thioester linkage of a 16-carbon palmitate lipid to an intracellular cysteine residue, is rapidly emerging as a fundamental, dynamic, and widespread post-translational mechanism to control the properties and function of ligand- and voltage-gated ion channels. Palmitoylation controls multiple stages in the ion channel life cycle, from maturation to trafficking and regulation. An emerging concept is that palmitoylation is an important determinant of channel regulation by other signaling pathways. The elucidation of enzymes controlling palmitoylation and developments in proteomics tools now promise to revolutionize our understanding of this fundamental post-translational mechanism in regulating ion channel physiology.
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http://dx.doi.org/10.1074/jbc.R110.210005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058972PMC
March 2011

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.

J Biol Chem 2011 Apr 5;286(14):11929-36. Epub 2011 Jan 5.

College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.

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.
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http://dx.doi.org/10.1074/jbc.M110.189779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069395PMC
April 2011

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

J Biol Chem 2010 Oct 6;285(43):33307-33314. Epub 2010 Aug 6.

From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M110.153940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963414PMC
October 2010

Multiple palmitoyltransferases are required for palmitoylation-dependent regulation of large conductance calcium- and voltage-activated potassium channels.

J Biol Chem 2010 Jul 27;285(31):23954-62. Epub 2010 May 27.

Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, University of Edinburgh, Edinburgh EH89XD, Scotland, United Kingdom.

Palmitoylation is emerging as an important and dynamic regulator of ion channel function; however, the specificity with which the large family of acyl palmitoyltransferases (zinc finger Asp-His-His-Cys type-containing acyl palmitoyltransferase (DHHCs)) control channel palmitoylation is poorly understood. We have previously demonstrated that the alternatively spliced stress-regulated exon (STREX) variant of the intracellular C-terminal domain of the large conductance calcium- and voltage-activated potassium (BK) channels is palmitoylated and targets the STREX domain to the plasma membrane. Using a combined imaging, biochemical, and functional approach coupled with loss-of-function (small interfering RNA knockdown of endogenous DHHCs) and gain-of-function (overexpression of recombinant DHHCs) assays, we demonstrate that multiple DHHCs control palmitoylation of the C terminus of STREX channels, the association of the STREX domain with the plasma membrane, and functional channel regulation. Cysteine residues 12 and 13 within the STREX insert were the only endogenously palmitoylated residues in the entire C terminus of the STREX channel. Palmitoylation of this dicysteine motif was controlled by DHHCs 3, 5, 7, 9, and 17, although DHHC17 showed the greatest specificity for this site upon overexpression of the cognate DHHC. DHHCs that palmitoylated the channel also co-assembled with the channel in co-immunoprecipitation experiments, and knockdown of any of these DHHCs blocked regulation of the channel by protein kinase A-dependent phosphorylation. Taken together our data reveal that a subset of DHHCs controls STREX palmitoylation and function and suggest that DHHC17 may preferentially target cysteine-rich domains. Finally, our approach may prove useful in elucidating the specificity of DHHC palmitoylation of intracellular domains of other ion channels and transmembrane proteins.
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http://dx.doi.org/10.1074/jbc.M110.137802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911306PMC
July 2010

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.

J Biol Chem 2010 Jul 17;285(30):23265-75. Epub 2010 May 17.

Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom.

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.
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http://dx.doi.org/10.1074/jbc.M110.139758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906319PMC
July 2010

A role for potassium permeability in the recognition, clearance, and anti-inflammatory effects of apoptotic cells.

Mol Neurobiol 2010 Aug 28;42(1):17-24. Epub 2010 Apr 28.

MRC Center for Inflammation Research, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, EH164TJ, UK.

The benefits of programmed cell death by apoptosis are the safe and efficient clearance of damaged, infected, or surplus cells, primarily mediated by tissue-resident macrophages or tissue-infiltrating blood monocytes that differentiate into macrophages. Microglial cells are macrophages of the brain parenchyma, important immune surveillance cells that respond to various injuries and diseases of the brain. It is often stated that how a macrophage interacts with an apoptotic cell defines subsequent inflammatory responses, i.e., will engulfment be beneficial or detrimental for tissue repair, regeneration, and immunity. Our focus has been to better understand how macrophages discriminate between living and dying cells. Following our initial findings with platelet endothelial cell adhesion molecule (PECAM)-1, our studies have revealed a key role for potassium ion permeability in regulating integrin-dependent binding of apoptotic cells by macrophages and their subsequent response to proinflammatory stimuli. Specifically, apoptotic cells represent a depolarizing stimulus for macrophages where PECAM-1-mediated cell-cell interactions delay subsequent membrane repolarization. It is salient that potassium leak represents an early feature of cells destined to die by apoptosis that could trigger depolarization of macrophages that lie in close apposition. We speculate that how a tissue-resident macrophage responds to strong depolarizing stimuli has wider implications for inflammation and autoimmunity.
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http://dx.doi.org/10.1007/s12035-010-8127-3DOI Listing
August 2010

Inducible knockout mutagenesis reveals compensatory mechanisms elicited by constitutive BK channel deficiency in overactive murine bladder.

FEBS J 2009 Mar 12;276(6):1680-97. Epub 2008 Feb 12.

Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Tübingen, Germany.

The large-conductance, voltage-dependent and Ca(2+)-dependent K(+) (BK) channel links membrane depolarization and local increases in cytosolic free Ca(2+) to hyperpolarizing K(+) outward currents, thereby controlling smooth muscle contractility. Constitutive deletion of the BK channel in mice (BK(-/-)) leads to an overactive bladder associated with increased intravesical pressure and frequent micturition, which has been revealed to be a result of detrusor muscle hyperexcitability. Interestingly, time-dependent and smooth muscle-specific deletion of the BK channel (SM-BK(-/-)) caused a more severe phenotype than displayed by constitutive BK(-/-) mice, suggesting that compensatory pathways are active in the latter. In detrusor muscle of BK(-/-) but not SM-BK(-/-) mice, we found reduced L-type Ca(2+) current density and increased expression of cAMP kinase (protein kinase A; PKA), as compared with control mice. Increased expression of PKA in BK(-/-) mice was accompanied by enhanced beta-adrenoceptor/cAMP-mediated suppression of contractions by isoproterenol. This effect was attenuated by about 60-70% in SM-BK(-/-) mice. However, the Rp isomer of adenosine-3',5'-cyclic monophosphorothioate, a blocker of PKA, only partially inhibited enhanced cAMP signaling in BK(-/-) detrusor muscle, suggesting the existence of additional compensatory pathways. To this end, proteome analysis of BK(-/-) urinary bladder tissue was performed, and revealed additional compensatory regulated proteins. Thus, constitutive and inducible deletion of BK channel activity unmasks compensatory mechanisms that are relevant for urinary bladder relaxation.
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http://dx.doi.org/10.1111/j.1742-4658.2009.06900.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025950PMC
March 2009

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

Proc Natl Acad Sci U S A 2008 Dec 19;105(52):21006-11. Epub 2008 Dec 19.

Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH89XD, Untied Kingdom.

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.
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http://dx.doi.org/10.1073/pnas.0806700106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2605631PMC
December 2008
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