scholarly journals Reduced function of the glutathione S-transferase S1 suppresses behavioral hyperexcitability in Drosophila expressing a mutant voltage-gated sodium channel

2020 ◽  
Author(s):  
Hung-Lin Chen ◽  
Junko Kasuya ◽  
Patrick Lansdon ◽  
Garrett Kaas ◽  
Hanxi Tang ◽  
...  
Keyword(s):  
Action Potentials ◽  
Null Allele ◽  
Single Gene ◽  
Loss Of Function ◽  
Wild Type ◽  
Bioactive Lipids ◽  
Glutathione S Transferase ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Prostaglandin D Synthase

ABSTRACTVoltage-gated sodium (Nav) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Nav-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Nav-channel mutant, paraShu. Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses paraShu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Nav-channel mutants, paraGEFS+ and parabss. Notably, paraShu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that paraShu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a paraShu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of paraShu phenotypes.

scholarly journals Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in Drosophila Expressing Mutant Voltage-Gated Sodium Channels

2020 ◽  
Vol 10 (4) ◽  
pp. 1327-1340
Author(s):  
Hung-Lin Chen ◽  
Junko Kasuya ◽  
Patrick Lansdon ◽  
Garrett Kaas ◽  
Hanxi Tang ◽  
...  
Keyword(s):  
Null Allele ◽  
Single Gene ◽  
Loss Of Function ◽  
Bioactive Lipids ◽  
Glutathione S Transferase ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Link Type ◽  
Voltage Gated Sodium Channels ◽  
Prostaglandin D Synthase

Voltage-gated sodium (Nav) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Nav-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Nav-channel mutant, paraShu. Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses paraShu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Nav-channel mutants, paraGEFS+ and parabss. Notably, paraShu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that paraShu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a paraShu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of paraShu phenotypes.

Abstract 1073: Cardiac Voltage-gated Nav channel Na v 1.5 Requires An AnkyrinG-dependent Pathway For Targeting in Cardiomyocytes

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
John S Lowe ◽  
Oleg Palygin ◽  
Patrick Wright ◽  
Erwin Shibata ◽  
Peter Mohler
Keyword(s):  
Ion Channels ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Excitable Cells ◽  
Membrane Expression ◽  
Voltage Gated ◽  
Ankyrin G ◽  
Ankyrin B ◽  
Dependent Pathway

Membrane localization of ion channels is very important for normal function in excitable cells. In heart, voltage-gated Na + channels are necessary for the rapid upstroke of the cardiomyocyte action potential, and variants in SCN5A (encodes Na v 1.5) are associated with fatal arrhythmias. We have identified ankyrin family proteins as critical components for normal ion channel and transporter targeting in cardiomyocytes. Humans with ANK2 (encodes ankyrin-B) loss of function variants display abnormal cardiac phenotypes and risk for sudden cardiac death. Mice that lack ankyrin-B expression display a similar phenotype. Our most recent results demonstrate that a second ankyrin gene product, ankyrin-G (encoded by ANK 3) is critical for targeting Na v 1.5 to specific cardiomyocyte membrane domains. We assessed the hypothesis that Na v 1.5 membrane expression and localization is controlled by an ankyrin-G-dependent pathway and disruption of ankyrin-G/Na v 1.5 interactions lead to human cardiac disease in this study. We used a combination of techniques including biochemistry, confocal microscopy, lentiviral expression, and electrophysiology to evaluate the functional relationship between ankyrin-G and Na v 1.5. We defined the structural elements on ankyrin-G and Na v 1.5 for their interaction using site-directed mutagenesis and in vitro binding assays. Lentiviral expression of shRNA targeted to rat 190 kD ankyrin-G effectively reduced the expression of ankyrin-G with a concomitant reduction of Na v 1.5 in immunofluorescence and immunoblot assays. Further-more, primary cardiomyocytes with reduced ankyrin-G expression have a significant reduction in Na + current density with no evident biophysical effects on Ca 2+ current or inactivation-gating of Na v 1.5 These results confirm the importance of ankyrin polypeptides for normal cardiac function and shed new light on the importance of intracellular trafficking pathways for the delivery and stability of critical ion channels and transporters in excitable cells.

scholarly journals Functions of Presynaptic Voltage-gated Calcium Channels

Function ◽  
2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Annette C Dolphin
Keyword(s):  
Calcium Channels ◽  
Neurotransmitter Release ◽  
Action Potentials ◽  
Voltage Dependence ◽  
Kinetic Properties ◽  
Excitable Cells ◽  
Single Action ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channels ◽  
Vesicular Release

Abstract Voltage-gated calcium channels are the principal conduits for depolarization-mediated Ca2+ entry into excitable cells. In this review, the biophysical properties of the relevant members of this family of channels, those that are present in presynaptic terminals, will be discussed in relation to their function in mediating neurotransmitter release. Voltage-gated calcium channels have properties that ensure they are specialized for particular roles, for example, differences in their activation voltage threshold, their various kinetic properties, and their voltage-dependence of inactivation. All these attributes play into the ability of the various voltage-gated calcium channels to participate in different patterns of presynaptic vesicular release. These include synaptic transmission resulting from single action potentials, and longer-term changes mediated by bursts or trains of action potentials, as well as release resulting from graded changes in membrane potential in specialized sensory synapses.

scholarly journals Action potentials in Xenopus oocytes triggered by blue light

2020 ◽  
Vol 152 (5) ◽  
Author(s):  
Florian Walther ◽  
Dominic Feind ◽  
Christian vom Dahl ◽  
Christoph Emanuel Müller ◽  
Taulant Kukaj ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Blue Light ◽  
Action Potentials ◽  
Xenopus Oocytes ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Excitable Cells ◽  
Voltage Gated

Voltage-gated sodium (Na+) channels are responsible for the fast upstroke of the action potential of excitable cells. The different α subunits of Na+ channels respond to brief membrane depolarizations above a threshold level by undergoing conformational changes that result in the opening of the pore and a subsequent inward flux of Na+. Physiologically, these initial membrane depolarizations are caused by other ion channels that are activated by a variety of stimuli such as mechanical stretch, temperature changes, and various ligands. In the present study, we developed an optogenetic approach to activate Na+ channels and elicit action potentials in Xenopus laevis oocytes. All recordings were performed by the two-microelectrode technique. We first coupled channelrhodopsin-2 (ChR2), a light-sensitive ion channel of the green alga Chlamydomonas reinhardtii, to the auxiliary β1 subunit of voltage-gated Na+ channels. The resulting fusion construct, β1-ChR2, retained the ability to modulate Na+ channel kinetics and generate photosensitive inward currents. Stimulation of Xenopus oocytes coexpressing the skeletal muscle Na+ channel Nav1.4 and β1-ChR2 with 25-ms lasting blue-light pulses resulted in rapid alterations of the membrane potential strongly resembling typical action potentials of excitable cells. Blocking Nav1.4 with tetrodotoxin prevented the fast upstroke and the reversal of the membrane potential. Coexpression of the voltage-gated K+ channel Kv2.1 facilitated action potential repolarization considerably. Light-induced action potentials were also obtained by coexpressing β1-ChR2 with either the neuronal Na+ channel Nav1.2 or the cardiac-specific isoform Nav1.5. Potential applications of this novel optogenetic tool are discussed.

scholarly journals Coccidioides posadasii Contains a Single 1,3-β-Glucan Synthase Gene That Appears To Be Essential for Growth

2005 ◽  
Vol 4 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Ellen M. Kellner ◽  
Kris I. Orsborn ◽  
Erin M. Siegel ◽  
M. Alejandra Mandel ◽  
Marc J. Orbach ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Wall ◽  
Microscopic Examination ◽  
Null Allele ◽  
Single Gene ◽  
Protein Product ◽  
Wild Type ◽  
Coccidioides Posadasii ◽  
Glucan Synthase ◽  
High Degree

ABSTRACT 1,3-β-Glucan synthase is responsible for the synthesis of β-glucan, an essential cell wall structural component in most fungi. We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-β-glucan synthase. A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi. FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature. We used Agrobacterium-mediated transformation to create strains that harbor ΔFKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain. The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with ΔFKS1::hygB alleles revealed abnormal swelling of hyphal elements.

Mutations in sfdA and sfdB Suppress Multiple Developmental Mutations in Aspergillus nidulans

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 159-168
Author(s):  
Ellen M Kellner ◽  
Thomas H Adams
Keyword(s):  
Aspergillus Nidulans ◽  
Null Allele ◽  
Hyphal Growth ◽  
Loss Of Function ◽  
Wild Type ◽  
Regulatory Pathway ◽  
Submerged Cultures ◽  
Developmental Defects ◽  
Suppressor Mutations ◽  
Its Gene

Abstract Conidiophore morphogenesis in Aspergillus nidulans occurs in response to developmental signals that result in the activation of brlA, a well-characterized gene that encodes a transcription factor that is central to asexual development. Loss-of-function mutations in flbD and other fluffy loci have previously been shown to result in delayed development and reduced expression of brlA. flbD message is detectable during both hyphal growth and conidiation, and its gene product is similar to the Myb family of transcription factors. To further understand the regulatory pathway to brlA activation and conidiation, we isolated suppressor mutations that rescued development in strains with a flbD null allele. We describe here two new loci, designated sfdA and sfdB for suppressors of flbD, that bypass the requirement of flbD for development. sfd mutant alleles were found to restore developmental timing and brlA expression to strains with flbD deletions. In addition, sfd mutations suppress the developmental defects in strains harboring loss-of-function mutations in fluG, flbA, flbB, flbC, and flbE. All alleles of sfdA and sfdB that we have isolated are recessive to their wild-type alleles in diploids. Strains with mutant sfd alleles in otherwise developmentally wild-type backgrounds have reduced growth phenotypes and develop conidiophores in submerged cultures.

scholarly journals Role of sodium channel subtype in action potential generation by neocortical pyramidal neurons

2018 ◽  
Vol 115 (30) ◽  
pp. E7184-E7192 ◽  
Author(s):  
Efrat Katz ◽  
Ohad Stoler ◽  
Anja Scheller ◽  
Yana Khrapunsky ◽  
Sandra Goebbels ◽  
...  
Keyword(s):  
Action Potentials ◽  
Pyramidal Neurons ◽  
Sodium Current ◽  
Na Channel ◽  
Loss Of Function ◽  
Wild Type ◽  
Strong Reduction ◽  
Hyperpolarizing Shift ◽  
Neocortical Pyramidal Neurons

Neocortical pyramidal neurons express several distinct subtypes of voltage-gated Na+ channels. In mature cells, Nav1.6 is the dominant channel subtype in the axon initial segment (AIS) as well as in the nodes of Ranvier. Action potentials (APs) are initiated in the AIS, and it has been proposed that the high excitability of this region is related to the unique characteristics of the Nav1.6 channel. Knockout or loss-of-function mutation of the Scn8a gene is generally lethal early in life because of the importance of this subtype in noncortical regions of the nervous system. Using the Cre/loxP system, we selectively deleted Nav1.6 in excitatory neurons of the forebrain and characterized the excitability of Nav1.6-deficient layer 5 pyramidal neurons by patch-clamp and Na+ and Ca2+ imaging recordings. We now report that, in the absence of Nav1.6 expression, the AIS is occupied by Nav1.2 channels. However, APs are generated in the AIS, and differences in AP propagation to soma and dendrites are minimal. Moreover, the channels that are expressed in the AIS still show a clear hyperpolarizing shift in voltage dependence of activation, compared with somatic channels. The only major difference between Nav1.6-null and wild-type neurons was a strong reduction in persistent sodium current. We propose that the molecular environment of the AIS confers properties on whatever Na channel subtype is present and that some other benefit must be conferred by the selective axonal presence of the Nav1.6 channel.

The roles of the homeobox genes aristaless and Distal-less in patterning the legs and wings of Drosophila

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4483-4493 ◽  
Author(s):  
G. Campbell ◽  
A. Tomlinson
Keyword(s):  
Null Allele ◽  
Normal Development ◽  
Distal Tibia ◽  
Homeobox Genes ◽  
Clonal Analysis ◽  
Loss Of Function ◽  
Wild Type ◽  
Adhesive Properties ◽  
Wing Imaginal Discs ◽  
Mutant Cells

In the leg and wing imaginal discs of Drosophila, the expression domains of the homeobox genes aristaless (al) and Distal-less (Dll) are defined by the secreted signaling molecules Wingless (Wg) and Decapentaplegic (Dpp). Here, the roles played by al and Dll in patterning the legs and wings have been investigated through loss of function studies. In the developing leg, al is expressed at the presumptive tip and a molecularly defined null allele of al reveals that its only function in patterning the leg appears to be to direct the growth and differentiation of the structures at the tip. In contrast, Dll has previously been shown to be required for the development of all of the leg more distal than the coxa. Dll protein can be detected in a central domain in leg discs throughout most of larval development, and in mature discs this domain corresponds to the distal-most region of the leg, the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. However, earlier in development Dll is required in more proximal regions of the leg suggesting it is expressed at high levels in these cells early in development but not later. This reveals a correlation between a temporal requirement for Dll and position along the proximodistal axis; how this may relate to the generation of the P/D axis is discussed. Dll is required in the distal regions of the leg for the expression of tarsal-specific genes including al and bric-a-brac. Dll mutant cells in the leg sort out from wild-type cells suggesting one function of Dll here is to control adhesive properties of cells. Dll is also required for the normal development of the wing, primarily for the differentiation of the wing margin.

scholarly journals A Xenopus oocyte model system to study action potentials

2018 ◽  
Vol 150 (11) ◽  
pp. 1583-1593 ◽  
Author(s):  
Aaron Corbin-Leftwich ◽  
Hannah E. Small ◽  
Helen H. Robinson ◽  
Carlos A. Villalba-Galea ◽  
Linda M. Boland
Keyword(s):  
Action Potentials ◽  
Subcellular Location ◽  
Model System ◽  
K Channel ◽  
Functional Coupling ◽  
Xenopus Laevis Oocytes ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Channel Expression ◽  
Electrical Signaling

Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na+- and K+-selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the functional coupling of these proteins by membrane potential, make it challenging to understand the roles of different channels in initiating and “temporally shaping” the AP. Here, to address this problem, we focus our efforts on finding conditions that allow reliable AP recordings from Xenopus laevis oocytes coexpressing Na+ and K+ channels. As a proof of principle, we show how the expression of a variety of K+ channel subtypes can modulate excitability in this minimal model system. This approach raises the prospect of studies on the modulation of APs by pharmacological or biological means with a controlled background of Na+ and K+ channel expression.

Understanding Genotypes and Phenotypes of the Mutations in Voltage- Gated Sodium Channel α Subunits in Epilepsy

2019 ◽  
Vol 18 (4) ◽  
pp. 266-272 ◽  
Author(s):  
Yijun Feng ◽  
Shuzhang Zhang ◽  
Zhiping Zhang ◽  
Jingkang Guo ◽  
Zhiyong Tan ◽  
...  
Keyword(s):  
Action Potentials ◽  
Individualized Medicine ◽  
Genetic Alterations ◽  
Genetic Modifiers ◽  
Excitable Cells ◽  
Disease Mechanism ◽  
Α Subunit ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Α Subunits

Background & Objective: Voltage-gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in most excitable cells. In general, a VGSC consists of one pore-forming α subunit and two auxiliary β subunits. Genetic alterations in VGSCs genes, including both α and β subunits, are considered to be associated with epileptogenesis as well as seizures. This review aims to summarize the mutations in VGSC α subunits in epilepsy, particularly the pathophysiological and pharmacological properties of relevant VGSC mutants. Conclusion: The review of epilepsy-associated VGSC α subunits mutants may not only contribute to the understanding of disease mechanism and genetic modifiers, but also provide potential theoretical targets for the precision and individualized medicine for epilepsy.

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