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

Abstract

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...

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNa V 1.7

“…The inhibitory potency of HwTx-IV and two analogues on hNa V 1.7 was assessed using a calcium assay on a Fluorimetric Imaging Plate Reader (FLIPR TETRA ) assay as previously described [41]. SH-SY5Y cells expressing hNa V 1.7 were maintained in RPMI medium supplemented with 15% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, 100 units/mL streptomycin and 2 mM L-glutamine, with subculturing every 72 h (70-80% confluence).…”

“…Immediately prior to assays, media was removed and cells were washed in physiological salt solution (PSS: 140 mM NaCl, 11.5 mM glucose, 5.9 mM KCl, 1.4 mM MgCl 2 , 1.2 mM NaH 2 PO 4 , 5 mM NaHCO 3 , 1.8 mM CaCl 2 and 10 mM HEPES, pH 7.4). The SH-SY5Y cells were then incubated for 30 min at 37 °C with FLIPR calcium 4 dye, 30 nM of OD1 (a selective modulator of hNa V 1.7 inactivation), and 0.1% (w/v) bovine serum albumin (BSA) diluted in PSS buffer as previously described [41]. Peptides were prepared to achieve final concentrations ranging from 3 M to 1.7…”

δ-Conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails

“…Crude venom isolated from C. suturatus elicited increases in intracellular Ca 2þ that were associated with oscillations in SH-SY5Y cells and potentiated veratridine-induced Ca 2þ responses which are mediated through endogenously expressed hNa V 1.2, hNa V 1.3 and hNa V 1.7 [10]. Using assay-guided fractionation, we found that activity was associated with a single peak eluting at 63% B that was dominated by a single monoisotopic mass of A BLAST search using this sequence as a query revealed that Bt6.4, a superfamily O1 conotoxin isolated from the vermivorous Conus betulinus, was the closest homologue to this 27 residue long peptide (electronic supplementary material, figure S1), with 96% sequence identity (Asp .…”

“…(b) Activity-guided isolation of SuVIA Activity-guided isolation of SuVIA was carried out using a FLIPR high-throughput Ca 2þ assay as previously described [9,10]. Briefly, the human neuroblastoma cell line SH-SY5Y (ATCC) endogenously expressing hNa V 1.2, hNa V 1.3 and hNa V 1.7 [10] was cultured in Roswell Park Memorial Institute (RPMI) medium containing 15% fetal bovine serum (FBS) and 2 mM L-glutamine and split every 3-4 days using 0.25% trypsin/EDTA (Life Technologies, Mulgrave, Victoria, Australia).…”

“…(10), pH 7.4). To assess activation of Na V subtypes by SuVIA, a two-addition protocol (for each addition: read interval 1 s, number of reads 300) measuring changes in fluorescence (excitation 510 -545 nm, emission 565 -625 nm) in response to addition of varying concentrations of SuVIA and an EC 10 concentration of the site 2 toxin veratridine (10-20 mM) was used.…”