β-Subunits of voltage-gated sodium channels in human prostate cancer: quantitative in vitro and in vivo analyses of mRNA expression

Diss, James K.J.; Fraser, Scott P.; Walker, Marjorie M.; Patel, Anup; Latchman, David S.; Djamgoz, Mustafa B.A.

doi:10.1038/sj.pcan.4501012

Cited by 53 publications

(68 citation statements)

References 47 publications

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“…The cells were then transferred to the FLIPR TETRA+ fluorescent plate reader and changes in fluorescence (excitation 510-545 nm; emission 565-625 nm) in response to addition of agonists was measured every second for 300 s. designed using Primer BLAST, and human β subunit primers were as previously described in the literature [11] (see Table 1). Plasmids encoding for Nav1.1-1.8 and β1-β3 subunits verified amplification of the correct products for each subtype (data not shown).…”

Section: Fluorescence Measurement Of Membrane Potential Changesmentioning

confidence: 99%

“…Primer pairs used for PCR of Nav subtypes and β subunits. Human Nav primers were designed using Primer BLAST, and human β subunit primers were as previously described in the literature [11]. For each Nav subtype and β subunit, GenBank accession numbers, forward and reverse primer sequences, expected PCR product size and locations are listed.…”

Section: Statement Of Conflicts Of Interestmentioning

confidence: 99%

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Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Vetter

Mozar

Durek

et al. 2012

Biochemical Pharmacology

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Abbreviations: Nav, voltage-gated sodium channel; TTX, tetrodotoxin; ProTxII, β/ω-theraphotoxin-Tp2a; Ca 2+ , calcium ion; DMEM, Dulbecco's Modified Eagle's Medium; FBS, foetal bovine serum; PBS, phosphate buffered saline; PSS, physiological salt solution; HBS, HEPES-buffered saline; AFU, arbitrary fluorescence unit; SEM, standard error of the mean; Fluo-4 AM, Fluo-4 acetoxymethylester; RPMI, Roswell Park Memorial Institute; BSA, bovine serum albumin; CCD, charge-coupled device; DAPI, 4',6-diamidino-2-phenylindole 2 AbstractThe human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Nav modulators, but little is known about the pharmacology of their endogenously expressed Navs. The aim of this study was to determine the expression of endogenous Nav α and β subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Nav isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Nav isoforms including Nav1.2 and Nav1.7. Activation of endogenously expressed Navs with veratridine or the scorpion toxin OD1 caused membrane depolarization and subsequent Ca 2+ influx through voltage-gated L-and N-type calcium channels, allowing Nav activation to be detected with membrane potential and fluorescent Ca 2 dyes. -Conotoxin TIIIA and ProTxII identified Nav1.2 and Nav1.7 as the major contributors of this response. The Nav1.7-selective scorpion toxin OD1 in combination with veratridine produced a Nav1.7-selective response, confirming that endogenously expressed human Nav1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening.Key Words: SH-SY5Y; Ca 2+ ; Nav1.7; ProTxII; OD1 3 IntroductionVoltage-gated sodium channels (Nav) are complex transmembrane proteins comprised of a poreforming α subunit and accessory β subunits that play an essential role in the initiation and propagation of action potentials in excitable cells. To date, apart from the related Nax which appears to function as a sodium sensor [1,2], nine isoforms termed Nav1.1 -Nav1.9 have been functionally defined as sodium-selective ion channels [3]. Their distinct tissue distribution and amenability to modulation by toxins and drugs has led to significant interest in Nav as therapeutic targets in a number of poorly treated conditions ranging from epilepsy to cardiac arrhythmias and pain [4]. In recent years Nav1.7 has emerged as an attractive drug target, with expression restricted to a subset of nociceptive neurons that is expected to limit on-target side effects of pharmacological modulators of Nav1.7 [5]. In addition, loss-of-function mutations in humans have highlighted the possibility that Nav1.7 inhibition could produce complete loss of pain sensations without dose-limiting side effects [6]. However, it remains unclear if such an effect can be translated to the clinic...

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Section: Fluorescence Measurement Of Membrane Potential Changesmentioning

confidence: 99%

Section: Statement Of Conflicts Of Interestmentioning

confidence: 99%

Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Vetter

Mozar

Durek

et al. 2012

Biochemical Pharmacology

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“…Strikingly, the position of 58 Cys in β4 corresponds to that of the Cys ( 26 Cys) thought to be involved in linking β2 to Na v 1.1 (40). Here, removing the covalent bond with Na v 1.1 by mutating 26 Cys to Ala results in an altered subcellular localization pattern of β2. Given the prominent position of 58 Cys in the β4 crystal structure and the functional importance of the corresponding residue in β2, we asked whether 58 Cys plays a role in determining the influence of β4 on Na v 1.2 toxin pharmacology.…”

Section: Significancementioning

confidence: 99%

“…Of the four known β-subunits and their splice variants (15)(16)(17)(18)(19)(20), β4 is unique in that it enables resurgent current, a feature that renders certain Na v channel isoforms capable of high-frequency firing in excitable tissues (21). Moreover, aberrant behavior of the ubiquitously expressed β4 subunit has been implicated in long-QT syndrome (LQTS) (22), LQTS-associated Sudden Infant Death Syndrome (23), atrial fibrillation (24), Huntington's disease (25), and prostate cancer (26), possibly through dysregulation of the Na v channel signaling complex. β4 also is targeted by β-and γ-secretase enzymes from the amyloidogenic pathway, a recent observation that suggests a potential contribution of this particular subunit to the development of Alzheimer's disease (27).…”

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confidence: 99%

Crystallographic insights into sodium-channel modulation by the β4 subunit

Gilchrist

Das

Petegem

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

105

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Voltage-gated sodium (Na v ) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, β-subunits modify Na v channel function and cause debilitating disorders when mutated. While investigating whether β-subunits also influence ligand interactions, we found that β4 dramatically alters toxin binding to Na v 1.2. To explore these observations further, we solved the crystal structure of the extracellular β4 domain and identified 58 Cys as an exposed residue that, when mutated, eliminates the influence of β4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of β4. Disrupting this bridge by introducing a β1 mutation implicated in epilepsy repositions the 58 Cys-containing loop and disrupts β4 modulation of Na v 1.2. Overall, the principles emerging from this work (i) help explain tissuedependent variations in Na v channel pharmacology; (ii) enable the mechanistic interpretation of β-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant β-subunit behavior.voltage-gated sodium channel | beta4 subunit | ProTx-II | X-ray structure | disease mutations

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“…The voltage-gated sodium channels (VGSCs) potentiate breast cancer metastasis [341], and indeed the involvement of NaV in the galvanotaxis that allows prostate and breast cancer cells to move across vessel lumens [213,337,[343][344][345][346][347][348][349] is one of the leading stories on ion channels in cancer. Highly up-regulated activities of NaV confers on cancer cells directional motility and invasive characteristics via Ca 2+ and pH-sensitive cytoskeletal remodeling processes which facilitate metastasis [337,341].…”

Section: Ion Channels Are Oncogenes and Important Drug Targetsmentioning

confidence: 99%

Cancer as a disorder of patterning information: computational and biophysical perspectives on the cancer problem

Moore

Walker

Levin

2017

Converg. Sci. Phys. Oncol.

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TOPICAL REVIEWCancer as a disorder of patterning information: computational and biophysical perspectives on the cancer problem IntroductionCancer is well-recognized not only as an extremely pernicious medical problem, but also as a group of phenomena deeply connected to the fundamental questions of evolution, multicellularity, pattern (disregulation, and the interplay between the genome and the environment [1,2]. Two fundamental paradigms currently divide the field. One is that cancer results from disorders of genetics: cancer cells are fundamentally broken due to genomic mutation or instability, and their clonal progeny form tumors and metastases. This view is sometimes called the SMT, somatic mutation theory [3][4][5]. A competing perspective is that of cancer as a circuit disease-a disorder of the complex biophysical, transcriptional, and epigenetic dynamics that regulate cellular state and the ability of cells to cooperate in vivo [6][7][8]. Under this view (sometimes called the tissue organization field theory or TOFT, [9,10]), cancer is akin to a traffic jam [11]-the problem is not a permanent discrete alteration within a founder cell and all of its clonal descendants, but an undesirable stable attractor in the complex network of controls that normally guides anatomical homeostasis [12].The relative merits of the two models have been discussed extensively [10,[13][14][15][16][17] AbstractThe current paradigm views cancer as arising clonally from a degradation of genetic information in single cells. A complementary perspective, originating at the dawn of modern developmental biology, is that cancer is the result of a system disorder of algorithms that normally orchestrate individual cell activities toward specific anatomical structures and away from tumorigenesis. A view of cancer as a disease of geometry focuses on the pathways that allow cells to cooperate to build and maintain large-scale anatomical patterning. Cancer may result when cells stop maintaining higher-order structures and reduce the boundary of their computational selves to a single-cell level, reverting to a unicellular lifestyle in which the rest of the organism is merely part of the environment at the expense of which all living things survive. While this view has been widely discussed, little progress has been made in providing a quantitative, mechanistic framework within which this perspective's specific and unique implications for treatment strategies can be tested and biomedically exploited. Here, we highlight two recent areas of progress which may facilitate much-needed progress on the cancer problem. First, we review the roles that endogenous bioelectrical networks, operating across many tissues in vivo, play as a medium of information processing in tumor suppression, progression, and reprogramming. Second, we provide a primer to the development of computational theory and tools for quantifying the information and causal control structures in cancer and other complex biological systems. Rigorous mathematical formalisms now exist to...

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β-Subunits of voltage-gated sodium channels in human prostate cancer: quantitative in vitro and in vivo analyses of mRNA expression

Cited by 53 publications

References 47 publications

Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Crystallographic insights into sodium-channel modulation by the β4 subunit

Cancer as a disorder of patterning information: computational and biophysical perspectives on the cancer problem

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