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1.  A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder 
Human Molecular Genetics  2014;23(23):6395-6406.
We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G>A, p.Arg394His). KCNH7 c.1181G>A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G>A had the highest enrichment among individuals with bipolar spectrum disorder (χ2 = 7.3) and the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031) and any major affective disorder (P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.
doi:10.1093/hmg/ddu335
PMCID: PMC4222358  PMID: 24986916
2.  A point mutation in the human Slo1 channel that impairs its sensitivity to omega-3 docosahexaenoic acid 
The Journal of General Physiology  2013;142(5):507-522.
Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) at nanomolar concentrations reversibly activate human large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels containing auxiliary β1 or β4 subunits in cell-free patches. Here we examined the action of DHA on the Slo1 channel without any auxiliary subunit and sought to elucidate the biophysical mechanism and the molecular determinants of the DHA sensitivity. Measurements of ionic currents through human Slo1 (hSlo1) channels reveal that the stimulatory effect of DHA does not require activation of the voltage or Ca2+ sensors. Unlike gating of the hSlo1 channel, that of the Drosophila melanogaster Slo1 (dSlo1) channel is unaltered by DHA. Our mutagenesis study based on the differential responses of human and dSlo1 channels to DHA pinpoints that Y318 near the cytoplasmic end of S6 in the hSlo1 channel is a critical determinant of the stimulatory action of DHA. The mutation Y318S in hSlo1, which replaces Y with S as found in dSlo1, greatly diminishes the channel’s response to DHA with a 22-carbon chain whether β1 or β4 is absent or present. However, the responses to α-linolenic acid, an omegea-3 fatty acid with an 18-carbon chain, and to arachidonic acid, an omega-6 fatty acid with a 20-carbon chain, remain unaffected by the mutation. Y318 in the S6 segment of hSlo1 is thus an important determinant of the electrophysiological response of the channel to DHA. Furthermore, the mutation Y318S may prove to be useful in dissecting out the complex lipid-mediated modulation of Slo1 BK channels.
doi:10.1085/jgp.201311061
PMCID: PMC3813383  PMID: 24127525
3.  C-027 Inhibits IgE-mediated passive sensitization bronchoconstriction and acts as a histamine and serotonin antagonist in human airways 
Atopic asthma is poorly controlled by current therapies. Newer therapies and novel antihistamines are, therefore, required to treat patients whose atopic asthma is not controlled. For the first time, C-027 is shown to antagonize histamine, IgE-mediated and serotonin-induced contraction in human airways and vessels. Human precision-cut lung slices (PCLS, 250 µm thick), containing an airway or blood vessel, were pretreated with either C-027 (2 hours) or with vehicle alone and were contracted with histamine or serotonin. Known antihistamine was used as a comparator in antihistamine studies. Also, human airways were contracted via IgE passive sensitization in the presence or absence of C-027 or fexofenadine. Affinity of C-027 toward human G-protein coupled receptors was also determined, as well as the drug's biodistribution in murine model. C-027 was shown to have the highest affinity toward human histamine and serotonin receptors. Subsequently, C-027 was shown to antagonize histamine- and serotonin-induced airway and vascular smooth muscle contraction, respectively, and histamine-released bronchocontraction mediated by IgE passive sensitization in human small airways. C-027 also inhibited histamine-mediated single-cell calcium ion release. Low levels of C-027 were found in murine brain tissue. Collectively, these data suggest that C-027 markedly inhibits IgE-induced bronchoconstriction and antagonizes histamine and serotonin-contraction with little biodistribution in the brain. The compound may offer a future therapy for allergen-induced airway hyperresponsiveness in patients with asthma.
doi:10.2500/aap.2011.32.3460
PMCID: PMC3968313  PMID: 22195688
Airway smooth muscle; allergy; antagonist; anti-histamine; anti-serotonic; asthma; IgE mediated; novel compound; PCLS; reverse agonist
4.  Functional Consequences of Methionine Oxidation of hERG Potassium Channels 
Biochemical pharmacology  2007;74(5):702-711.
Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in hERG1 (human ether à go-go related gene 1), which encodes cardiac IKr channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300 µM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to −60 or −100 mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30 mV but accelerated during depolarization to 0 or −10 mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel.
doi:10.1016/j.bcp.2007.06.002
PMCID: PMC3905454  PMID: 17624316
hERG; K-channel; chloramine-T; methionine; oxidation; methionine sulfoxide reductase
6.  Cysteines control the N- and C-linker-dependent gating of KCNH1 potassium channels 
Biochimica et Biophysica Acta  2012;1818(5):1187-1195.
KCNH1 (EAG1) is a member of the Kv family of voltage-gated potassium channels. However, KCNH1 channels also show some amino-acid sequence similarity to cyclic-nucleotide-regulated channels: they harbor an N-terminal PAS domain, a C-terminal cyclic nucleotide binding homology domain (cNBHD), and N- and C-terminal binding sites for calmodulin. Another notable feature is the channels’ high sensitivity toward oxidative modification. Using human KCNH1 expressed in Xenopus oocytes and HEK 293 cells we investigated how oxidative modification alters channel function. Intracellular application of H2O2 or cysteine-specific modifiers potently inhibited KCNH1 channels in two phases. Our systematic cysteine mutagenesis study showed that the rapid and dominant phase was attributed to a right-shift in the voltage dependence of activation, caused by chemical modification of residues C145 and C214. The slow component depended on the C-terminal residues C532 and C562. The cysteine pairs are situated at structural elements linking the transmembrane S1 segment with the PAS domain (N-linker) and the transmembrane channel gate S6 with the cNBH domain (C-linker), respectively. The functional state of KCNH1 channels is determined by the oxidative status of these linkers that provide an additional dimension of channel regulation.
doi:10.1016/j.bbamem.2012.01.021
PMCID: PMC3319268  PMID: 22310694
Potassium channel; Reactive oxygen species; Oxidation; Sulfhydryl modification; Ether à go-go; Gating
7.  Trichostatin A Abrogates Airway Constriction, but Not Inflammation, in Murine and Human Asthma Models 
Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (RL) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca2+. The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in RL and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca2+ in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca2+ in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.
doi:10.1165/rcmb.2010-0276OC
PMCID: PMC3297166  PMID: 22298527
HDAC; asthma; allergen; mice; trichostatin A
8.  Drosophila QVR/SSS modulates the activation and C-type inactivation kinetics of Shaker K+ channels 
The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K+ currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, we demonstrated that SLEEPLESS (SSS) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. We show here that SSS accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this SSS-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. We also find that SSS decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss of function mutations. Kinetic modeling based on the in vitro results suggests that the SSS-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking SSS. Sleep duration in qvr/sss null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of SSS in the regulation of sleep in Drosophila correlates more strongly with the effects of SSS on Shaker kinetics than current magnitude.
doi:10.1523/JNEUROSCI.0502-11.2011
PMCID: PMC3183472  PMID: 21813698
9.  Where’s the gate? Gating in the deep pore of the BKCa channel 
The Journal of General Physiology  2011;138(2):133-136.
doi:10.1085/jgp.201110681
PMCID: PMC3149440  PMID: 21746844
10.  An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma 
PLoS ONE  2012;7(1):e28504.
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
doi:10.1371/journal.pone.0028504
PMCID: PMC3257220  PMID: 22253691
11.  Bilirubin oxidation end products (BOXes) directly alter K+ channels important in regulation of vascular tone 
The exact etiology of delayed cerebral vasospasm following cerebral hemorrhage is not clear but a family of compounds termed bilirubin oxidation end products (BOXes) derived from heme has been implicated. Because proper regulation of vascular smooth muscle tone involves large-conductance Ca2+- and voltage-dependent Slo1 K+ (BK, maxiK, KCa1.1) channels, we examined whether BOXes altered functional properties of the channel. Electrophysiological measurements of Slo1 channels heterologously expressed in a human cell line as well as of native mouse BK channels in isolated cerebral myocytes showed that BOXes markedly diminished open probability. Biophysically BOXes specifically stabilized the conformations of the channel with its ion conduction gate closed. The results of chemical amino-acid modifications and molecular mutagenesis together suggest that two specific lysine residues in the structural element linking the transmembrane ion-permeation domain to the carboxyl cytosolic domain of the Slo1 channel are critical in determining sensitivity of the channel to BOXes. Inhibition of Slo1 BK channels by BOXes may contribute to development of delayed cerebral vasospasm following brain hemorrhage.
doi:10.1038/jcbfm.2010.54
PMCID: PMC2970662  PMID: 20424637
Slo1 BK channels; Ca2+-dependent K+ channels; Maxi-K channels; vasoconstriction; bilirubin
12.  Bilirubin oxidation end products directly alter K+ channels important in the regulation of vascular tone 
The exact etiology of delayed cerebral vasospasm following cerebral hemorrhage is not clear, but a family of compounds termed ‘bilirubin oxidation end products (BOXes)' derived from heme has been implicated. As proper regulation of vascular smooth muscle tone involves large-conductance Ca2+- and voltage-dependent Slo1 K+ (BK, maxiK, KCa1.1) channels, we examined whether BOXes altered functional properties of the channel. Electrophysiological measurements of Slo1 channels heterologously expressed in a human cell line and of native mouse BK channels in isolated cerebral myocytes showed that BOXes markedly diminished open probability. Biophysically, BOXes specifically stabilized the conformations of the channel with its ion conduction gate closed. The results of chemical amino-acid modifications and molecular mutagenesis together suggest that two specific lysine residues in the structural element linking the transmembrane ion-permeation domain to the carboxyl cytosolic domain of the Slo1 channel are critical in determining the sensitivity of the channel to BOXes. Inhibition of Slo1 BK channels by BOXes may contribute to the development of delayed cerebral vasospasm following brain hemorrhage.
doi:10.1038/jcbfm.2010.54
PMCID: PMC2970662  PMID: 20424637
bilirubin; Ca2+-dependent K+ channels; maxi-K channels; Slo1 BK channels; vasoconstriction
13.  Distinct Functions of Glial and Neuronal Dystroglycan in the Developing and Adult Mouse Brain 
SUMMARY
Cobblestone (type II) lissencephaly and mental retardation are characteristic features of a subset of congenital muscular dystrophies that include Walker-Warburg Syndrome, Muscle-Eye-Brain disease, and Fukuyama-type congenital muscular dystrophy. Although the majority of clinical cases are genetically undefined, several causative genes have been identified that encode known or putative glycosyltransferases in the biosynthetic pathway of dystroglycan. Here we test the effects of brain-specific deletion of dystroglycan, and show distinct functions for neuronal and glial dystroglycan. Deletion of dystroglycan in the whole brain produced glial/neuronal heterotopia resembling the cerebral cortex malformation in cobblestone lissencephaly. In wild-type mice, dystroglycan stabilizes the basement membrane of the glia limitans, thereby supporting the cortical infrastructure necessary for neuronal migration. This function depends on extracellular dystroglycan interactions, since the cerebral cortex developed normally in transgenic mice that lack the dystroglycan intracellular domain. Also, forebrain histogenesis was preserved in mice with neuron-specific deletion of dystroglycan, but hippocampal long-term potentiation was blunted, as is also the case in the Largemyd mouse, in which dystroglycan glycosylation is disrupted. Our findings provide genetic evidence that neuronal dystroglycan plays a role in synaptic plasticity and that glial dystroglycan is involved in forebrain development. Differences in dystroglycan glycosylation in distinct cell types of the CNS may therefore contribute to the diversity of dystroglycan function in the CNS, as well as to the broad clinical spectrum of type II lissencephalies.
doi:10.1523/JNEUROSCI.3247-10.2010
PMCID: PMC2979314  PMID: 20980614
14.  A mutually exclusive alternative exon of slo1 codes for a neuronal BK channel with altered function 
Channels (Austin, Tex.)  2008;2(4):278-282.
Large-conductance Ca2+- and voltage-activated K+ (BK) channels are comprised of four pore-forming α-subunits (Slo1), whose mRNA is alternatively spliced in a cell-specific manner. Here we report the first case of a correctly spliced mutually exclusive exon in a mammalian BK channel; an exon coding for the region from S6 to the RCK1 domain is exchanged for an alternative exon of the same length. The Slo1 transcript with this novel exon is present in native brain tissues and inclusion of the alternative exon profoundly alters the channel’s gating characteristics: faster activation at low Ca2+ concentrations and greater open probability at resting membrane potential at high Ca2+ concentrations. The novel gating features conferred by the alternative exon are dominant over those of the commonly described Slo1 variant when coexpressed. These data show that evolutionary preserved regulation of alternative Slo1 splicing of the S6-RCK1 linker creates fine-tuning of neuronal excitability.
PMCID: PMC2921853  PMID: 18719396
Slo1; BK channels; gene splicing; Ca2+ dependence; K(Ca2+); KCNMA1
15.  Modulation of BKCa channel gating by endogenous signaling molecules 
Physiology (Bethesda, Md.)  2009;24:26-35.
Large-conductance Ca2+- and voltage-activated K+ (BKCa, MaxiK or Slo1) channels are expressed in almost every tissue in our body and participate in many critical functions such as neuronal excitability, vascular tone regulation and neurotransmitter release. The functional versatility of BKCa channels owes in part to the availability of a spectacularly wide array of biological modulators of the channel function. In this review, we focus on modulation of BKCa channels by small endogenous molecules, emphasizing their molecular mechanisms. The mechanistic information available from studies on the small naturally occurring modulators is expected to contribute to our understanding of the physiological and pathophysiological roles of BKCa channels.
doi:10.1152/physiol.00032.2008
PMCID: PMC2914466  PMID: 19196649
16.  Oxidation of multiple methionine residues impairs rapid sodium channel inactivation 
Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.
doi:10.1007/s00424-008-0477-6
PMCID: PMC2913308  PMID: 18369661
Sodium channel; Na+ channel; Inactivation; Methionine oxidation; Chloramine-T; Lucifer Yellow; Patch clamp
17.  Comparative effects of H+ and Ca2+ on large-conductance Ca2+- and voltage-gated Slo1 K+ channels 
Channels (Austin, Tex.)  2009;3(4):249-258.
Summary
Large-conductance Ca2+- and voltage-gated Slo1 BK channels are allosterically activated by depolarization and intracellular ligands such as Ca2+. Of the two high-affinity Ca2+ sensors present in the channel, the RCK1 sensor also mediates H+-dependent activation of the channel. In this study, we examined the comparative mechanisms of the channel activation by Ca2+ and H+. Steady-state macroscopic conductance-voltage measurements as well as single-channel openings at negative voltages where voltage-sensor activation is negligible showed that at respective saturating concentrations Ca2+ is more effective in relative stabilization of the open conformation than H+. Calculations using the Debye-Hückel formulation suggest that small structural changes in the RCK1 sensor, on the order of few angstroms, may accompany the H+-mediated opening of the channel. While the efficacy of H+ in activation of the channel is less than that of Ca2+, H+ more effectively accelerates the activation kinetics when examined at the concentrations equipotent on macroscopic voltage-dependent activation. The RCK1 sensor therefore is capable of transducing the nature of the ligand bound and transmits qualitatively different information to the channel’s permeation gate.
PMCID: PMC2824562  PMID: 19617704
BK channel; Slo1 channel; Ca2+-dependent K+ channel; voltage-gated channel; calcium; potassium; proton
18.  Reciprocal regulation of the Ca2+ and H+ sensitivity in the Slo1 BK channel conferred by the RCK1 domain 
Increasing evidence suggests that intracellular H+ directly stimulates large-conductance Ca2+- and voltage-activated K+ (Slo1 BK) channels, thus providing a crucial link between membrane excitability and cell metabolism. Here we report that two histidine residues, His365 and His394, located in the intracellular RCK1 domain serve as the H+ sensors of the Slo1 BK channel. Activation of the channel by H+ requires electrostatic interactions between the histidine residues and a nearby negatively charged residue involved in the channel’s high-affinity Ca2+ sensitivity. Reciprocally, His365 and His394 also participate in the Ca2+-dependent activation of the channel, functioning as Ca2+ mimetics once protonated. Therefore, a common motif in the RCK1 domain mediates the stimulatory effects of both H+ and Ca2+, and provides a basis for the bidirectional coupling of cell metabolism and membrane electrical excitability.
doi:10.1038/nsmb.1398
PMCID: PMC2905141  PMID: 18345016
19.  SLEEPLESS, a Ly–6/Neurotoxin Family Member, Regulates Levels, Localization, and Activity of Shaker 
Nature neuroscience  2009;13(1):69-75.
Sleep is a whole–organism phenomenon accompanied by global changes in neural activity. We previously identified SLEEPLESS (SSS) as a novel glycosylphosphatidyl–inositol–anchored protein required for sleep in Drosophila. Here, we demonstrate a critical role for SSS in regulating the sleep–modulating potassium channel, Shaker. SSS and Shaker exhibit similar expression patterns in the brain and specifically affect each other’s expression levels. sss mutants exhibit altered Shaker localization, reduced Shaker current density, and slower Shaker current kinetics. Transgenic expression of sss in sss mutants rescues defects in Shaker expression and activity cell–autonomously and also suggests that SSS functions in wake–promoting, cholinergic neurons. Importantly, in heterologous cells, SSS accelerates kinetics of Shaker currents and can be co–immunoprecipitated with Shaker, suggesting that SSS interacts with Shaker and modulates its activity. SSS is predicted to belong to the Ly–6/neurotoxin superfamily, suggesting a novel mechanism for regulation of neuronal excitability by endogenous toxin–like molecules.
doi:10.1038/nn.2454
PMCID: PMC2842941  PMID: 20010822
20.  Differential trafficking of carboxyl isoforms of Ca2+-gated (Slo1) potassium channels 
FEBS letters  2007;581(5):1000-1008.
The pore-forming subunit of the large-conductance Ca2+-dependent K+ (Slo1) channel is encoded by one gene. However, the functional properties of Slo1 channels are diverse in part because of their numerous regulatory mechanisms including posttranslational modification and alternative splicing. In particular, multiple splice variants of the pore-forming subunit have been reported but their significance is only beginning to be elucidated. Here we examined the cell biological properties of the three common C-terminal isoforms that differ in the last 8 (Slo1_ERL and Slo1_VYR) or 61 residues (Slo1_DEC). We found that Slo1_DEC, the longest isoform, shows dramatically reduced surface expression compared to that of Slo1_ERL or Slo1_VYR. Immunocytochemistry revealed that a large fraction of Slo1_DEC remains localized in endoplasmic reticulum (ER). Using a GST fusion protein containing the Slo1_DEC-specific sequence, affinity purification was carried out to isolate interacting proteins. The identified proteins include protein phosphatases 2A (PP2A-A), , actin, and tubulin. The PP2A-A interaction is specific to Slo1_DEC and causes a significant reduction of phosphorylation in Slo1_DEC but not Slo1_ERL or Slo1_VYR. The results together support the notion that Slo1_DEC nucleates isoform-specific protein complexes and possesses a cis element(s) for regulating trafficking of the Slo1 channels.
doi:10.1016/j.febslet.2007.01.077
PMCID: PMC1896267  PMID: 17303127
21.  Oxidative damage, aging and anti-aging strategies 
Age  2005;27(3):183-199.
The last two decades brought remarkable insight into the nature of normal aging in multicellular organisms. However, we are still far away from realizing extension of maximum lifespan in humans. An important modulator of lifespan is oxidative damage induced by reactive species, such as reactive oxygen species (ROS). Studies from yeast, Caenorhabditis and Drosophila primarily focused on (1) reduced generation or (2) elimination of ROS but have two principal shortcomings: (1) dietary restriction and single gene mutations are often associated with physiological impairments and (2) overexpression of components of the antioxidant system extend lifetime only under stress-induced conditions. Recent results from Drosophila indicate the involvement of an endogenous repair and elimination system for oxidatively damaged proteins in the process of aging. This system includes methionine sulfoxide reductase A (MSRA) and the carbonyl reductase Sniffer, the protein-ubiquitin ligase Parkin and the chaperone Hsp22. In this review we summarize different anti-aging strategies and discuss a synergistic interaction between protection against free radicals and specific repair/elimination of oxidative damage in lifespan extension primarily using the model system Drosophila. To achieve lifespan extension, available experiments are often methodically grouped into (1) caloric restriction, (2) single gene mutation, and (3) overexpression of genes. Here we summarize different strategies by a more causal classification: (1) prevention of ROS generation, (2) reducing free ROS level, and (3) repair and elimination of ROS-damaged proteins.
doi:10.1007/s11357-005-2915-0
PMCID: PMC3458494  PMID: 23598652
antioxidant system; Drosophila; methionine sulfoxide reductases; oxidative stress theory of aging; protein oxidation; ROS
22.  Heme Regulates Allosteric Activation of the Slo1 BK Channel 
Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state.
doi:10.1085/jgp.200509262
PMCID: PMC2266614  PMID: 15955873
23.  The β1 Subunit Enhances Oxidative Regulation of Large-Conductance Calcium-activated K+ Channels 
The Journal of General Physiology  2004;124(4):357-370.
Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) α subunits alone. Because native BKCa channel complexes may include the auxiliary subunit β1, we investigated whether β1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with β1 present shifted the half-activation voltage much further in the hyperpolarizing direction (−75 mV) as compared with that with α alone (−30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of β1. Oxidation of cysteine and methionine residues within β1 was not involved in these potentiated effects because expression of mutant β1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type β1. Unlike the results with α alone, oxidation by Ch-T caused a significant acceleration of channel activation only when β1 was present. The β1 M177 mutation disrupted normal channel activation and prevented the Ch-T–induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming α subunit are greatly amplified by the presence of β1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within β1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with β1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.
doi:10.1085/jgp.200409144
PMCID: PMC2233902  PMID: 15452197
BKCa; hSlo; chloramine-T; methionine; cysteine
24.  Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels 
The Journal of General Physiology  2001;117(3):253-274.
Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.
PMCID: PMC2225619  PMID: 11222629
chloramine-T; methionine; methionine sulfoxide; methionine sulfoxide reductase; cysteine
25.  Dihydropyridine Action on Voltage-dependent Potassium Channels Expressed in Xenopus Oocytes  
The Journal of General Physiology  1997;109(2):169-180.
Dihydropyridines (DHPs) are well known for their effects on L-type voltage-dependent Ca2+ channels. However, these drugs also affect other voltage-dependent ion channels, including Shaker K+ channels. We examined the effects of DHPs on the Shaker K+ channels expressed in Xenopus oocytes. Intracellular applications of DHPs quickly and reversibly induced apparent inactivation in the Shaker K+ mutant channels with disrupted N- and C-type inactivation. We found that DHPs interact with the open state of the channel as evidenced by the decreased mean open time. The DHPs effects are voltage-dependent, becoming more effective with hyperpolarization. A model which involves binding of two DHP molecules to the channel is consistent with the results obtained in our experiments.
PMCID: PMC2220064  PMID: 9041446
potassium channels; dihydropyridines

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