scholarly journals Regulation of Nav1.6 and Nav1.8 peripheral nerve Na+ channels by auxiliary β-subunits

2011 ◽  
Vol 106 (2) ◽  
pp. 608-619 ◽  
Author(s):  
Juan Zhao ◽  
Michael E. O'Leary ◽  
Mohamed Chahine
Keyword(s):  
Current Density ◽  
Small Diameter ◽  
Fold Increase ◽  
Β Subunit ◽  
Drg Neurons ◽  
Α Subunit ◽  
Terminal Domain ◽  
Steady State Inactivation ◽  
Hyperpolarizing Shift ◽  
Auxiliary Β Subunits

Voltage-gated Na+ (Nav) channels are composed of a pore-forming α-subunit and one or more auxiliary β-subunits. The present study investigated the regulation by the β-subunit of two Na+ channels (Nav1.6 and Nav1.8) expressed in dorsal root ganglion (DRG) neurons. Single cell RT-PCR was used to show that Nav1.8, Nav1.6, and β1–β3 subunits were widely expressed in individually harvested small-diameter DRG neurons. Coexpression experiments were used to assess the regulation of Nav1.6 and Nav1.8 by β-subunits. The β1-subunit induced a 2.3-fold increase in Na+ current density and hyperpolarizing shifts in the activation (−4 mV) and steady-state inactivation (−4.7 mV) of heterologously expressed Nav1.8 channels. The β4-subunit caused more pronounced shifts in activation (−16.7 mV) and inactivation (−9.3 mV) but did not alter the current density of cells expressing Nav1.8 channels. The β3-subunit did not alter Nav1.8 gating but significantly reduced the current density by 31%. This contrasted with Nav1.6, where the β-subunits were relatively weak regulators of channel function. One notable exception was the β4-subunit, which induced a hyperpolarizing shift in activation (−7.6 mV) but no change in the inactivation or current density of Nav1.6. The β-subunits differentially regulated the expression and gating of Nav1.8 and Nav1.6. To further investigate the underlying regulatory mechanism, β-subunit chimeras containing portions of the strongly regulating β1-subunit and the weakly regulating β2-subunit were generated. Chimeras retaining the COOH-terminal domain of the β1-subunit produced hyperpolarizing shifts in gating and increased the current density of Nav1.8, similar to that observed for wild-type β1-subunits. The intracellular COOH-terminal domain of the β1-subunit appeared to play an essential role in the regulation of Nav1.8 expression and gating.

Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits

1997 ◽  
Vol 273 (6) ◽  
pp. C1889-C1899 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Albert Tousson ◽  
Dale J. Benos
Keyword(s):  
Fluorescence Intensity ◽  
Expression System ◽  
Fold Increase ◽  
Na Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Animal Pole ◽  
Α Subunit ◽  
Amino Acid Peptide ◽  
Xenopus Oocyte Expression

Using the Xenopus oocyte expression system, we examined the mechanisms by which the β- and γ-subunits of an epithelial Na+channel (ENaC) regulate α-subunit channel activity and the mechanisms by which β-subunit truncations cause ENaC activation. Expression of α-ENaC alone produced small amiloride-sensitive currents (−43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of β- and γ-ENaC to −1,476 ± 254 nA ( n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the α-subunit of ENaC. Truncation of the last 75 amino acids of the β-subunit COOH terminus, as found in the original pedigree of individuals with Liddle’s syndrome, caused a 4.4-fold ( n = 17) increase of the amiloride-sensitive currents compared with wild-type αβγ-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human β-ENaC significantly reduced the amiloride-sensitive currents by 40–50%. These observations suggest a tonic inhibitory role on the channel’s open probability ( P o) by the COOH terminus of β-ENaC. We conclude that the changes of current observed with coexpression of the β- and γ-subunits or those observed with β-subunit truncation are likely the result of changes of channel density in combination with large changes of P o.

Delayed rectifier K+ current of dog bronchial myocytes: effect of pollen sensitization and PKC activation

1998 ◽  
Vol 275 (2) ◽  
pp. L336-L347 ◽  
Author(s):  
Gareth J. Waldron ◽  
Stefan B. Sigurdsson ◽  
Ernesto A. Aiello ◽  
Andrew J. Halayko ◽  
Newman L. Stephens ◽  
...  
Keyword(s):  
Current Density ◽  
Voltage Dependence ◽  
Ragweed Pollen ◽  
Delayed Rectifier ◽  
Airway Smooth Muscle Cells ◽  
Average Current Density ◽  
Whole Cell Patch Clamp ◽  
Steady State Inactivation ◽  
Pkc Activation ◽  
Hyperpolarizing Shift

The properties of delayed rectifier K+ current [ I K(dr)] of canine airway smooth muscle cells isolated from small bronchi and its modulation by protein kinase C (PKC) were studied by whole cell patch clamp. I K(dr) activated positive to −40 mV, with half-maximal activation at −16 ± 1.2 mV ( n = 15) and average current density of 31 ± 2.6 pA/pF ( n = 15) at +30 mV. The capacitive surface area, current density, and voltage dependence of activation of I K(dr) of myocytes of ragweed pollen-sensitized dogs were not different from age-matched control dogs. However, the sensitization reduced the availability of I K(dr) between −40 and −20 mV due to a hyperpolarizing shift in the voltage dependence of steady-state inactivation (−29.9 ± 1.2 in sensitized versus −26.0 ± 0.7 mV in control dogs, n = 9 and 11, respectively; P < 0.05). PKC activation with diacylglycerol analog or phorbol ester depressed I K(dr) amplitude, whereas an inactive diacylglycerol analog had no effect. The hyperpolarizing shift in voltage dependence of inactivation and/or modulation of I K(dr) by PKC may be two mechanisms that contribute to the enhanced reactivity of bronchial tissues from ragweed pollen-sensitized dogs.

Neonatal colonic inflammation sensitizes voltage-gated Na+ channels via upregulation of cystathionine β-synthetase expression in rat primary sensory neurons

2013 ◽  
Vol 304 (9) ◽  
pp. G763-G772 ◽  
Author(s):  
Ruobing Qu ◽  
Jingde Tao ◽  
Yongmeng Wang ◽  
Youlang Zhou ◽  
Geping Wu ◽  
...  
Keyword(s):  
Current Density ◽  
Visceral Pain ◽  
Fold Increase ◽  
Male Rats ◽  
Dependent Manner ◽  
Drg Neurons ◽  
Visceral Hyperalgesia ◽  
Colonic Inflammation ◽  
Voltage Gated ◽  
Enhanced Expression

The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood, and treatment remains difficult. We have previously reported that colon-specific dorsal root ganglion (DRG) neurons were hyperactive in a rat model of IBS induced by neonatal colonic inflammation (NCI). This study was designed to examine plasticity of voltage-gated Na+ channel activities and roles for the endogenous hydrogen sulfide-producing enzyme cystathionine β-synthetase (CBS) in chronic visceral hyperalgesia. Abdominal withdrawal reflex (AWR) scores were recorded in response to graded colorectal distention in adult male rats as a measure of visceral hypersensitivity. Colon-specific DRG neurons were labeled with 1,1′-dioleyl-3,3,3′,3-tetramethylindocarbocyanine methanesulfonate and acutely dissociated for measuring Na+ channel currents. Western blot analysis was employed to detect changes in expressions of voltage-gated Na+ (NaV) channel subtype 1.7, NaV1.8, and CBS. NCI significantly increased AWR scores when compared with age-matched controls. NCI also led to an ∼2.5-fold increase in Na+ current density in colon-specific DRG neurons. Furthermore, NCI dramatically enhanced expression of NaV1.7, NaV1.8, and CBS in colon-related DRGs. CBS was colocalized with NaV1.7 or -1.8 in colon-specific DRG neurons. Administration of O-(carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, remarkably suppressed Na+ current density and reduced expression of NaV1.7 and NaV1.8. More importantly, intraperitoneal or intrathecal application of AOAA attenuated AWR scores in NCI rats in a dose-dependent manner. These data suggest that NCI enhances Na+ channel activity of colon DRG neurons, which is most likely mediated by upregulation of CBS expression, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

scholarly journals C-terminal domain small phosphatase 1 (CTDSP1) regulates growth factor expression and axonal regeneration in peripheral nerve tissue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Noreen M. Gervasi ◽  
Alexander Dimtchev ◽  
Desraj M. Clark ◽  
Marvin Dingle ◽  
Alexander V. Pisarchik ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Peripheral Nerve ◽  
Neurotrophic Factor ◽  
Axon Growth ◽  
Nerve Tissue ◽  
Mesenchymal Progenitor Cells ◽  
Drg Neurons ◽  
Peripheral Neurons ◽  
Terminal Domain ◽  
Factor Expression

AbstractPeripheral Nerve Injury (PNI) represents a major clinical and economic burden. Despite the ability of peripheral neurons to regenerate their axons after an injury, patients are often left with motor and/or sensory disability and may develop chronic pain. Successful regeneration and target organ reinnervation require comprehensive transcriptional changes in both injured neurons and support cells located at the site of injury. The expression of most of the genes required for axon growth and guidance and for synapsis formation is repressed by a single master transcriptional regulator, the Repressor Element 1 Silencing Transcription factor (REST). Sustained increase of REST levels after injury inhibits axon regeneration and leads to chronic pain. As targeting of transcription factors is challenging, we tested whether modulation of REST activity could be achieved through knockdown of carboxy-terminal domain small phosphatase 1 (CTDSP1), the enzyme that stabilizes REST by preventing its targeting to the proteasome. To test whether knockdown of CTDSP1 promotes neurotrophic factor expression in both support cells located at the site of injury and in peripheral neurons, we transfected mesenchymal progenitor cells (MPCs), a type of support cells that are present at high concentrations at the site of injury, and dorsal root ganglion (DRG) neurons with REST or CTDSP1 specific siRNA. We quantified neurotrophic factor expression by RT-qPCR and Western blot, and brain-derived neurotrophic factor (BDNF) release in the cell culture medium by ELISA, and we measured neurite outgrowth of DRG neurons in culture. Our results show that CTDSP1 knockdown promotes neurotrophic factor expression in both DRG neurons and the support cells MPCs, and promotes DRG neuron regeneration. Therapeutics targeting CTDSP1 activity may, therefore, represent a novel epigenetic strategy to promote peripheral nerve regeneration after PNI by promoting the regenerative program repressed by injury-induced increased levels of REST in both neurons and support cells.

The Stability Enhancement of Nitrile Hydratase from Bordetella petrii by Swapping the C-terminal Domain of β subunit

2015 ◽  
Vol 178 (8) ◽  
pp. 1481-1487 ◽  
Author(s):  
Weifeng Sun ◽  
Longbao Zhu ◽  
Xianggui Chen ◽  
Lunjie Wu ◽  
Zhemin Zhou ◽  
...  
Keyword(s):  
Nitrile Hydratase ◽  
Β Subunit ◽  
Terminal Domain ◽  
The Stability ◽  
Stability Enhancement

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Thyroid hormone regulation of mRNAs encoding thyrotropin β-subunit, glycoprotein α-subunit, and thyroid hormone receptors α and β in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas

2009 ◽  
Vol 202 (1) ◽  
pp. 43-54 ◽  
Author(s):  
Sean C Lema ◽  
Jon T Dickey ◽  
Irvin R Schultz ◽  
Penny Swanson
Keyword(s):  
Thyroid Hormone ◽  
Pituitary Gland ◽  
Teleost Fish ◽  
Hormone Receptors ◽  
Pimephales Promelas ◽  
Early Gene ◽  
Thyroid Hormone Receptors ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3′-triiodo-l-thyronine (T3) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T3 elevated circulating total T3, while methimazole depressed plasma levels of total thyroxine (T4). In both sexes, this methimazole-induced reduction in T4 led to elevated mRNA abundance for thyrotropin β-subunit (tshβ) in the pituitary gland. Fish treated with T3 had elevated transcript levels for TR isoforms α and β (trα and trβ) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T3 elevated gene transcripts for tshβ, glycoprotein hormone α-subunit (gphα), and trβ, while not affecting trα levels. Taken together, these results demonstrate negative feedback of T4 on pituitary tshβ, identify trα and trβ as T3-autoinduced genes in the brain and liver, and provide new evidence that tshβ, gphα, and trβ are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

scholarly journals Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

2017 ◽  
Vol 474 (5) ◽  
pp. 751-769 ◽  
Author(s):  
M. Kristian Koski ◽  
Jothi Anantharajan ◽  
Petri Kursula ◽  
Prathusha Dhavala ◽  
Abhinandan V. Murthy ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Coiled Coil ◽  
Β Subunit ◽  
Dimerization Domain ◽  
Α Subunit ◽  
Protein Dimer ◽  
Symmetric Molecule ◽  
Asymmetric Shape ◽  
Proline Rich Peptide ◽  
Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

scholarly journals Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

1999 ◽  
Vol 337 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Emma C. LAW ◽  
Nigel J. SAVERY ◽  
Stephen J. W. BUSBY
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Class I ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Α Subunit ◽  
Terminal Domain ◽  
Up Elements ◽  
Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

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