The Experts below are selected from a list of 1059 Experts worldwide ranked by ideXlab platform
Kevin T Obyrne - One of the best experts on this subject based on the ideXlab platform.
-
urocortin 3 in the posterodorsal medial amygdala mediates psychosocial stress induced suppression of lh pulsatility in female mice
Endocrinology, 2021Co-Authors: Deyana Ivanova, Caitlin Mcintyre, Yali Liu, Lingsi Kong, Kevin T ObyrneAbstract:Psychosocial stress disrupts reproduction and interferes with pulsatile LH secretion. The posterodorsal medial amygdala (MePD) is an upstream modulator of the reproductive axis and stress. Corticotropin-releasing factor type-2 receptors (CRFR2) are activated in the presence of psychosocial stress together with increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in the MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress on LH pulsatility. Firstly, we administered Ucn3 into the MePD and monitored the effect on LH pulses in ovariectomised mice. Next, we delivered Astressin2B, a selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-Trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected Ucn3-cre-tdTomato mice with inhibitory DREADDs targeting MePD Ucn3 neurons while exposing mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone release. Administration of Ucn3 into the MePD dose-dependently inhibited LH pulses and administration of Astressin2B blocked the suppressive effect of TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses and corticosterone release. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and corticosterone secretion. Ucn3 signalling in the MePD plays a role in modulating the hypothalamic-pituitary-ganadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal centre in the interaction between the reproductive and stress axes.
-
urocortin 3 in the posterodorsal medial amygdala mediates psychosocial stress induced suppression of lh pulsatility in female mice
bioRxiv, 2021Co-Authors: Deyana Ivanova, Caitlin Mcintyre, Yali Liu, Lingsi Kong, Kevin T ObyrneAbstract:Exposure to psychosocial stress disrupts reproductive function and interferes with pulsatile luteinising hormone (LH) secretion in mammals. The posterodorsal sub-nucleus of the medial amygdala (MePD) is part of the limbic brain and is an upstream modulator of the reproductive axis as well as stress and anxiety states. Corticotropin releasing factor type-2 receptors (CRFR2) are activated in the presence of psychosocial stress together with an increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress exposure on LH pulsatility. Firstly, we administered Ucn3 into the MePD and monitored the effect on pulsatile LH secretion in ovariectomised mice. Next, we delivered Astressin2B, a highly selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-Trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected ovariectomised Ucn3-cre-tdTomato mice with inhibitory DREADDs targeting the MePD Ucn3 neurons while exposing the mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone (CORT) release. Administration of Ucn3 into the MePD dose-dependently inhibited pulsatile LH secretion and intra-MePD administration of Astressin2B blocked the suppressive effect TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses as well as CORT release in the presence of TMT. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and psychosocial stress-induced CORT secretion. Ucn3 signalling in the MePD plays a fundamental role in modulating the hypothalamic-pituitary-ganadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal centre in the crosstalk between the reproductive and stress axes.
-
kisspeptin neurones in the posterodorsal medial amygdala modulate sexual partner preference and anxiety in male mice
Journal of Neuroendocrinology, 2018Co-Authors: Daniel Adekunbi, Geffen Lass, K Shetty, O A Adegoke, Shelhwa Yeo, William H Colledge, Stafford L Lightman, Kevin T ObyrneAbstract:The posterodorsal medial amygdala (MePD) is a neural site in the limbic brain involved in regulating emotional and sexual behaviours. There is, however, limited information available on the specific neuronal cell type in the MePD functionally mediating these behaviours in rodents. The recent discovery of a significant kisspeptin neurone population in the MePD has raised interest in the possible role of kisspeptin and its cognate receptor in sexual behaviour. The present study therefore tested the hypothesis that the MePD kisspeptin neurone population is involved in regulating attraction towards opposite sex conspecifics, sexual behaviour, social interaction and the anxiety response by selectively stimulating these neurones using the novel pharmacosynthetic DREADDs (designer receptors exclusively activated by designer drugs) technique. Adult male Kiss-Cre mice received bilateral stereotaxic injections of a stimulatory DREADD viral construct (AAV-hSyn-DIO-hM3 D(Gq)-mCherry) targeted to the MePD, with subsequent activation by i.p. injection of clozapine-N-oxide (CNO). Socio-sexual behaviours were assessed in a counter-balanced fashion after i.p. injection of either saline or CNO (5 mg kg-1 ). Selective activation of MePD kisspeptin neurones by CNO significantly increased the time spent by male mice in investigating an oestrous female, as well as the duration of social interaction. Additionally, after CNO injection, the mice appeared less anxious, as indicated by a longer exploratory time in the open arms of the elevated plus maze. However, levels of copulatory behaviour were comparable between CNO and saline-treated controls. These data indicate that DREADD-induced activation of MePD kisspeptin neurones enhances both sexual partner preference in males and social interaction and also decreases anxiety, suggesting a key role played by MePD kisspeptin in sexual motivation and social behaviour.
-
kisspeptin in the medial amygdala and sexual behavior in male rats
Neuroscience Letters, 2016Co-Authors: Rebecca Gresham, Daniel Adekunbi, Shengyun Li, Minghan Hu, Xiao Feng Li, Kevin T ObyrneAbstract:The medial amygdala (MeA) is crucial for sexual behavior; kisspeptin (Kiss1) also plays a role in sexual function. Kisspeptin receptor (Kiss1r) knockout mice display no sexual behavior. Recently Kiss1 and Kiss1r have been discovered in the posterodorsal subnucleus of the medial amygdala (MePD). We hypothesised that Kiss1 in the MePD may have an influence on male sexual behavior. To test this we bilaterally cannulated the MePD and infused kisspeptin-10 in male rats. This caused the rats to have multiple erections, an effect specific to Kiss1 receptor activation, because Kiss1r antagonism blocked the erectile response. When Kiss1 was infused into the lateral cerebroventricle, there were no observed erections. We also measured the plasma levels of LH when Kiss1 is infused into the MePD or lateral cerebroventricle; Kiss1 increased plasma LH to comparable levels when infused into both sites. We conclude that Kiss1 has a role in male sexual behavior, which is specific to the MePD.
Yuesheng Zhang - One of the best experts on this subject based on the ideXlab platform.
-
abstract 552 pepd is an essential regulator of p53 tumor suppressor
Cancer Research, 2018Co-Authors: Lu Yang, Arup Bhattacharya, Yuesheng ZhangAbstract:Peptidase D (PEPD), also known as prolidase among other names, is an enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxy terminus and is believed to be important for collagen metabolism, as proline and hydroxyproline are very abundant in collagen. Interestingly, we recently found that eliminating cellular PEPD causes cell death and tumor regression due to p53 activation. Here, we show that PEPD binds to and suppresses over half of nuclear and cytoplasmic p53 under normal conditions, independent of its enzymatic activity. PEPD binds to the proline-rich domain in p53, which inhibits phosphorylation of nuclear p53 and MDM2-mediated mitochondrial translocation of nuclear and cytoplasmic p53. Indeed, PEPD competes with MDM2 for p53 binding. However, the PEPD-p53 complex is critical for p53 response to stress, as stress signals doxorubicin (DOX) and hydrogen peroxide each must free p53 from PEPD in order to achieve robust p53 activation, which is mediated by reactive oxygen species. Thus, PEPD stores p53 for stress response, but this also renders cells dependent on PEPD for survival as it suppresses p53. Our results reveal a major p53 regulatory mechanism and a critical physiological function of PEPD. The p53-PEPD system likely operates in most if not all cells, since both p53 and PEPD are expressed ubiquitously. Disrupting PEPD suppression of p53 may be an important therapeutic strategy in cancer, as our data show that PEPD knockdown by RNA interference in tumors in mice causes p53 activation in the tumor tissues and tumor regression. Our study also reveals a previously unrecognized anticancer mechanism of DOX. We show that the key step in DOX-induced p53 activation is the disruption of p53 association with PEPD via reactive oxygen species. This finding also raises the intriguing question of whether other stress-inducing anticancer agents also disrupt the PEPD-p53 complex for p53 activation and suggests that antioxidants may inhibit the anticancer activity of DOX and other agents by inhibiting p53 separation from PEPD. This work is supported by NCI grants and Roswell Park Alliance Foundation Grants. Citation Format: Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. PEPD is an essential regulator of p53 tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 552.
-
pepd is a pivotal regulator of p53 tumor suppressor
Nature Communications, 2017Co-Authors: Lu Yang, Arup Bhattacharya, Yuesheng ZhangAbstract:p53 tumor suppressor responds to various cellular stresses and regulates cell fate. Here, we show that peptidase D (PEPD) binds and suppresses over half of nuclear and cytoplasmic p53 under normal conditions, independent of its enzymatic activity. Eliminating PEPD causes cell death and tumor regression due to p53 activation. PEPD binds to the proline-rich domain in p53, which inhibits phosphorylation of nuclear p53 and MDM2-mediated mitochondrial translocation of nuclear and cytoplasmic p53. However, the PEPD-p53 complex is critical for p53 response to stress, as stress signals doxorubicin and H2O2 each must free p53 from PEPD in order to achieve robust p53 activation, which is mediated by reactive oxygen species. Thus, PEPD stores p53 for the stress response, but this also renders cells dependent on PEPD for survival, as it suppresses p53. This finding provides further understanding of p53 regulation and may have significant implications for the treatment of cancer and other diseases.
-
abstract p6 07 04 targeting erbb2 with human pepd
Cancer Research, 2015Co-Authors: Lu Yang, Arup Bhattacharya, Yuesheng ZhangAbstract:ErbB2, also known as Her2 or Neu, belongs to the ErbB family of plasma membrane-bound receptor tyrosine kinases, which also include ErbB1, ErbB3 and ErbB4. ErbB2 is best known for its involvement in human breast cancer. ErbB2 gene amplification occurs in ∼20% of breast cancer, and ErbB2 amplification or overexpression is a strong predictor of poor disease prognosis. ErbB2-targeted therapies, particularly humanized monoclonal antibody trastuzumab (Ttzm) in combination with chemotherapy, have shown considerable clinical efficacy. However, primary and secondary resistance remains a clinical challenge, and Ttzm, produced in mammalian cells, is very expensive. We have found that human prolidase, also known as peptidase D (PEPD) among several other names, binds to ErbB2 with high affinity (Kd = ∼7 nM) and binds as a homodimer (493 amino acids per subunit) to subdomain 3 in the extracellular domain of ErbB2. Each monomer of PEPD binds to one copy of ErbB2. However, PEPD is a weak ErbB1 binder (Kd = ∼5 μM) and does not bind to ErbB3 or ErbB4. PEPD is the first-ever natural ligand of ErbB2, and unlike the other ligands of ErbB receptors, it is devoid of an EGF motif. PEPD has been long known to hydrolyze dipeptides with proline or hydroxylproline at the carboxy terminus, but the dipeptidase activity of PEPD is not involved in ErbB2/ErbB1 modulation. In cells overexpressing ErbB2, where both activated dimers and inactive monomers of ErbB2 exist, as ErbB2 overexpression causes spontaneous dimerization, auto-tyrosine phosphorylation and recruitment and activation of downstream signals, PEPD rapidly binds to ErbB2 homodimers ( ∼30 min), but this binding causes ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. PEPD binding to ErbB2 subsequently causes pronounced ErbB2 depletion, resulting from its internalization and degradation. PEPD also strongly inhibits the DNA synthesis, anchorage-independent growth and invasion of cells that overexpress ErbB2, but has no effect on cells without overexpression of ErbB2. In fact, cells become sensitized to inhibition by PEPD upon achieving stable ErbB2 overexpression. Thus, the overall impact of PEPD on ErbB2 is inhibitory, and PEPD targets cells addicted to ErbB2. In ErbB2-overexpressing cells, at equimolar concentrations, PEPD was more effective than Ttzm in driving ErbB2 depletion, but is weaker than Ttzm in stimulating ErbB2 phosphorylation. In mouse tumor models, PEPD administered by intraperitoneal injection (Monday, Wednesday, Friday) at 0.2-2 mg/kg body weight strongly inhibited the growth of ErbB2-overexpressing tumors, but had no impact on tumors without ErbB2 overexpression, and the PEPD-treated mice showed no adverse effects. Given that the findings described above were made using human PEPD generated in bacteria, there is a distinct possibility that recombinant human PEPD may be a low cost alternative to Ttzm. Further investigation of the antitumor activity of PEPD and its modulation of ErbB2 signaling is warranted. Citation Format: Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. Targeting ErbB2 with human PEPD [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-04.
Maria Pia Longhese - One of the best experts on this subject based on the ideXlab platform.
-
Rif2 attenuates MRX function in end-tethering and NHEJ.
2016Co-Authors: Corinne Cassani, Elisa Gobbini, Michela Clerici, Weibin Wang, Hengyao Niu, Patrick Sung, Maria Pia LongheseAbstract:(A, B) DSB end-tethering. The assay was performed as described in Fig 4F and 4G. Plotted values are the mean value with error bars denoting s.d. (n = 3). (C) Plasmid re-ligation assay. The assay was done as described in Fig 6F. (D–F) Exponentially growing cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin, or MMS at the indicated concentrations.
-
DSB resection and end-tethering.
2016Co-Authors: Corinne Cassani, Elisa Gobbini, Michela Clerici, Weibin Wang, Hengyao Niu, Patrick Sung, Maria Pia LongheseAbstract:(A) Exponentially growing cell cultures were incubated with phleomycin (15 μg/ml) at time zero, followed by western blot analysis with anti-Rad53 antibodies. (B) Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and then released in YEPD containing 0.015% MMS, followed by western blot analysis with anti-Rad53 antibodies. (C) Exponentially growing YEPR cell cultures were transferred to YEPRG at time zero to induce HO that catalyzes an irreparable DSB at the MAT locus. Protein extracts were analyzed by western blot analysis with anti-Rad53 antibodies. (D) DSB resection. YEPR exponentially growing cell cultures were arrested in G2 with nocodazole and transferred to YEPRG in the presence of nocodazole at time zero. SspI-digested genomic DNA separated on alkaline agarose gel was hybridized with a single-stranded MAT probe that anneals with the unresected strand. Resection of the 5′ DNA end progressively eliminates SspI sites, producing larger SspI fragments (r1 through r7) detected by the probe. (E) Exponentially growing cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin, or MMS. (F, G) DSB end-tethering. Exponentially growing YEPR cell cultures were arrested in G2 with nocodazole (F) or in G1 with α-factor (G) at time zero and transferred to YEPRG in the presence of nocodazole or α-factor, respectively. Two-hundred cells for each strain were analyzed to determine the percentage of cells showing two LacI-GFP foci. (H) Sister chromatid cohesion. Exponentially growing YEPD cell cultures were arrested in G2 with nocodazole for 3 h to determine the percentage of cells showing two LacI-GFP foci. (I) Exponentially growing YEPR cell cultures, transformed with plasmid containing a galactose-inducible HO gene, were arrested in G2 with nocodazole at time zero and transferred to YEPRG in the presence of nocodazole for 2 h to determine the percentage of cells showing separated LacI-YFP and TetR-RFP foci. In all graphs, plotted values are the mean value with error bars denoting s.d. (n = 3).
-
rad50-V1269M tel1Δ mutant cells are defective in DSB repair by HR.
2016Co-Authors: Corinne Cassani, Elisa Gobbini, Michela Clerici, Weibin Wang, Hengyao Niu, Patrick Sung, Maria Pia LongheseAbstract:(A) System to detect ectopic recombination. HO generates a DSB at a MATa DNA sequence inserted on chromosome V, while the homologous MATa-inc region on chromosome III cannot be cut by HO and is used as a donor for HR-mediated repair, which can generate both noncrossover (NCO) and crossover (CO) products. E, EcoRI. (B) Exponentially growing YEPR cell cultures were transferred to YEPRG at time zero. Southern blot analysis of EcoRI-digested genomic DNA with the MATa probe depicted in A. (C) Densitometric analysis of CO versus NCO repair bands at the indicated times after HO induction (see Materials and Methods). (D) Mating type switching. Exponentially growing YEPR MATa cell cultures (raf) were transferred to YEPRG to induce HO. After 30 min (gal), cells were transferred to YEPD to allow mating type switching. StyI-BamHI-digested genomic DNA prepared at the indicated times after glucose addition was subjected to Southern blot analysis with a MATa probe. (E) Densitometric analysis of the MATα product band signals (see Materials and Methods). Plotted values are the mean value with error bars denoting s.d. (n = 3). * indicates cross hybridization signals.
-
The Tel1-kd variant restores DNA damage resistance and SSA in sae2Δ cells.
2015Co-Authors: Elisa Gobbini, Matteo Villa, Marco Gnugnoli, Luca Menin, Michela Clerici, Maria Pia LongheseAbstract:(A) Exponentially growing cells were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin or MMS. (B) DSB repair by SSA. The analysis was performed as described in Fig 4A. (C) Densitometric analysis of the product band signals. The experiment as in (B) was independently repeated three times and the mean values are represented with error bars denoting s.d. (n = 3). (D) Exponentially growing cells were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin or MMS.
-
Rad53-H88Y and Tel1-N2021D suppress the hypersensitivity to genotoxic agents of sae2Δ cells.
2015Co-Authors: Elisa Gobbini, Matteo Villa, Marco Gnugnoli, Luca Menin, Michela Clerici, Maria Pia LongheseAbstract:(A-D) Exponentially growing cells were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin or MMS.
Deyana Ivanova - One of the best experts on this subject based on the ideXlab platform.
-
urocortin 3 in the posterodorsal medial amygdala mediates psychosocial stress induced suppression of lh pulsatility in female mice
Endocrinology, 2021Co-Authors: Deyana Ivanova, Caitlin Mcintyre, Yali Liu, Lingsi Kong, Kevin T ObyrneAbstract:Psychosocial stress disrupts reproduction and interferes with pulsatile LH secretion. The posterodorsal medial amygdala (MePD) is an upstream modulator of the reproductive axis and stress. Corticotropin-releasing factor type-2 receptors (CRFR2) are activated in the presence of psychosocial stress together with increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in the MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress on LH pulsatility. Firstly, we administered Ucn3 into the MePD and monitored the effect on LH pulses in ovariectomised mice. Next, we delivered Astressin2B, a selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-Trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected Ucn3-cre-tdTomato mice with inhibitory DREADDs targeting MePD Ucn3 neurons while exposing mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone release. Administration of Ucn3 into the MePD dose-dependently inhibited LH pulses and administration of Astressin2B blocked the suppressive effect of TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses and corticosterone release. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and corticosterone secretion. Ucn3 signalling in the MePD plays a role in modulating the hypothalamic-pituitary-ganadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal centre in the interaction between the reproductive and stress axes.
-
urocortin 3 in the posterodorsal medial amygdala mediates psychosocial stress induced suppression of lh pulsatility in female mice
bioRxiv, 2021Co-Authors: Deyana Ivanova, Caitlin Mcintyre, Yali Liu, Lingsi Kong, Kevin T ObyrneAbstract:Exposure to psychosocial stress disrupts reproductive function and interferes with pulsatile luteinising hormone (LH) secretion in mammals. The posterodorsal sub-nucleus of the medial amygdala (MePD) is part of the limbic brain and is an upstream modulator of the reproductive axis as well as stress and anxiety states. Corticotropin releasing factor type-2 receptors (CRFR2) are activated in the presence of psychosocial stress together with an increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress exposure on LH pulsatility. Firstly, we administered Ucn3 into the MePD and monitored the effect on pulsatile LH secretion in ovariectomised mice. Next, we delivered Astressin2B, a highly selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-Trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected ovariectomised Ucn3-cre-tdTomato mice with inhibitory DREADDs targeting the MePD Ucn3 neurons while exposing the mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone (CORT) release. Administration of Ucn3 into the MePD dose-dependently inhibited pulsatile LH secretion and intra-MePD administration of Astressin2B blocked the suppressive effect TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses as well as CORT release in the presence of TMT. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and psychosocial stress-induced CORT secretion. Ucn3 signalling in the MePD plays a fundamental role in modulating the hypothalamic-pituitary-ganadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal centre in the crosstalk between the reproductive and stress axes.
James O Jackson - One of the best experts on this subject based on the ideXlab platform.
-
alternative splicing of na v 1 7 exon 5 increases the impact of the painful pepd mutant channel i1461t
Channels, 2009Co-Authors: Brian W Jarecki, Patrick L Sheets, Yucheng Xiao, James O JacksonAbstract:Alternative splicing is known to alter pharmacological sensitivities, kinetics, channel distribution under pathological conditions, and developmental regulation of VGSCs. Mutations that alter channel properties in Na(V)1.7 have been genetically implicated in patients with bouts of extreme pain classified as inherited erythromelalgia (IEM) or paroxysmal extreme pain disorder (PEPD). Furthermore, patients with IEM or PEPD report differential age onsets. A recent study reported that alternative splicing of Na(V)1.7 exon 5 affects ramp current properties. Since IEM and PEPD mutations also alter Na(V)1.7 ramp current properties we speculated that alternative splicing might impact the functional consequences of IEM or PEPD mutations. We compared the effects alternative splicing has on the biophysical properties of Na(V)1.7 wild-type, IEM (I136V) and PEPD (I1461T) channels. Our major findings demonstrate that although the 5A splice variant of the IEM channel had no functional impact, the 5A splice variant of the PEPD channel significantly hyperpolarized the activation curve, slowed deactivation and closed-state inactivation, shifted the ramp current activation to more hyperpolarized potentials, and increased ramp current amplitude. We hypothesize a D1/S3-S4 charged residue difference between the 5N (Asn) and the 5A (Asp) variants within the coding region of exon 5 may contribute to shifts in channel activation and deactivation. Taken together, the additive effects observed on ramp currents from exon 5 splicing and the PEPD mutation (I1461T) are likely to impact the disease phenotype and may offer insight into how alternative splicing may affect specific intramolecular interactions critical for voltage-dependent gating.
-
paroxysmal extreme pain disorder mutations within the d3 s4 s5 linker of nav1 7 cause moderate destabilization of fast inactivation
The Journal of Physiology, 2008Co-Authors: Brian W Jarecki, Patrick L Sheets, James O JacksonAbstract:Single-point missense mutations in the peripheral neuronal voltage-gated sodium channel Nav1.7 are implicated in the painful inherited neuropathy paroxysmal extreme pain disorder (PEPD). The Nav1.7 PEPD mutations are located in regions of the channel suggested to play important roles in fast inactivation. PEPD mutations in the putative inactivation gate have been reported to significantly impair fast inactivation, resulting in pronounced persistent currents. However, PEPD mutations in the S4–S5 linker of domain 3 (D3/S4–S5) had not been characterized and the roles of specific residues in this linker in channel gating are unclear. We functionally characterized two of the D3/S4–S5 PEPD mutations (V1298F and V1299F) and compared their effects on gating to an adjacent non-PEPD mutation (V1300F) and the I1461T PEPD mutation, located in the putative inactivation gate. The primary effect of the V1298F and V1299F mutations is to shift the voltage dependence of fast inactivation by ∼20 mV in the depolarizing direction. We observed a similar effect with the PEPD mutation I1461T. Interestingly, while all three PEPD mutations increased persistent currents, the relative amplitudes (∼6% of peak) were much smaller than previously reported for the I1461T mutation. In contrast, the main effect of the V1300F mutation was a depolarizing shift in the voltage dependence of activation. These data demonstrate that (1) mutations within D3/S4–S5 affect inactivation of Nav1.7 in a residue-specific manner and (2) disruption of the fast-inactivated state by PEPD mutations can be more moderate than previously indicated, which has important implications for the pathophysiology of PEPD.
