Site specific deacylation by ABHD17a controls BK channel splice variant activity

S-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 S-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 S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-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

Cell patterning on photolithographically defined parylene-C:SiO2 substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO(2 )wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO(2) regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology

The physiology of protein S-acylation

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

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

This is the final version of the article. Available from Wiley via the DOI in this record.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.P.J.D. was supported by an MRC PhD studentship in the College of Medicine and Veterinary Medicine, University of Edinburgh. Work was supported by grants to M.J.S. and P.R. from the Wellcome Trust (082407), to M.J.S. from MRC (J008893), and to R.B. and J.T. from the National Institutes of Health (DK43200)

Increased large conductance calcium-activated potassium (BK) channel expression accompanied by STREX variant downregulation in the developing mouse CNS

BACKGROUND: Large conductance calcium- and voltage activated potassium (BK) channels are important determinants of neuronal excitability through effects on action potential duration, frequency and synaptic efficacy. The pore- forming subunits are encoded by a single gene, KCNMA1, which undergoes extensive alternative pre mRNA splicing. Different splice variants can confer distinct properties on BK channels. For example, insertion of the 58 amino acid stress-regulated exon (STREX) insert, that is conserved throughout vertebrate evolution, encodes channels with distinct calcium sensitivity and regulation by diverse signalling pathways compared to the insertless (ZERO) variant. Thus, expression of distinct splice variants may allow cells to differentially shape their electrical properties during development. However, whether differential splicing of BK channel variants occurs during development of the mammalian CNS has not been examined. RESULTS: Using quantitative real-time polymerase chain reaction (RT-PCR) Taqman™ assays, we demonstrate that total BK channel transcripts are up regulated throughout the murine CNS during embryonic and postnatal development with regional variation in transcript levels. This upregulation is associated with a decrease in STREX variant mRNA expression and an upregulation in ZERO variant expression. CONCLUSION: As BK channel splice variants encode channels with distinct functional properties the switch in splicing from the STREX phenotype to ZERO phenotype during embryonic and postnatal CNS development may provide a mechanism to allow BK channels to control distinct functions at different times of mammalian brain development

Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C

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

Screening for functional neurological disorders by questionnaire

Objective: Diagnostic screening for functional neurological disorders (FNDs) continues to pose a challenge. Simple symptom counts fail clearly to discriminate patients with FND but there is increasing recognition of ‘positive’ features which are useful diagnostically during face-to-face assessments. A self-completed questionnaire evaluating specific features of FNDs would be useful for screening purposes in clinical and research settings. Methods: The Edinburgh Neurosymptoms Questionnaire (ENS) is a 30-item survey of presence and nature of: blackouts, weakness, hemisensory syndrome, memory problems, tremor, pain, fatigue, globus, multiple medical problems, and operations constructed via literature review and expert consensus. We conducted a pilot of the ENS on new general neurology clinic attendees at a large regional neuroscience centre. Patients were grouped according to consultant neurologist impression as having symptoms that were ‘Not at all’, 'somewhat’, ’Largely’ or ’Completely’ due to a functional disorder. Results: Blackouts, weakness and memory questions provided reasonable diagnostic utility (AUROC = 0.94, 0.71, 0.74 respectively) in single symptom analysis. All other symptoms lacked discriminating features. A multivariate linear model with all symptoms predicted functional classification with moderate diagnostic utility (AUROC = 0.83), specificity of 0.97, sensitivity of 0.47. Pain and blackout scores provided the most accurate predictor of functional classification. Conclusion: The ENS questionnaire provides some utility in differentiating patients presenting with functional blackouts but failed to provide diagnostic value in other types of FND, highlighting the limitations of this self-report tool