Cryptic prokaryotic promoters explain instability of recombinant neuronal sodium channels in bacteria

Mutations in genes encoding the human-brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, other neurodevelopmental disorders. A major obstacle hindering investigations functional consequences brain NaV channel mutations is an unexplained instability corresponding recombinant complementary DNA (cDNA) when propagated commonly used bacterial strains manifested by high spontaneous rates mutation. Here, using combination silico analysis, random site-directed mutagenesis, we investigated cause for NaV1.1 cDNA. We identified nucleotide sequences within coding region that resemble prokaryotic promoter-like elements, which presumed to drive transcription translationally toxic mRNAs bacteria as instability. further demonstrated disrupting these elements mitigate Extending observations, generated full-length plasmids one or two introns interrupt latent reading frames along minimum number silent changes achieved stable propagation bacteria. Expression stabilized cultured mammalian cells resulted properties matched their parental constructs. Our findings explain widely observed neuronal channels, describe re-engineered attenuate this problem. Recombinant brain-expressed important tools investigating structure–function relationships, drug discovery, determining naturally occurring neurological disorders such disorder (1Decaen P.G. George Jr., A.L. Thompson C.H. Sodium channelopathies central nervous system.in: Bhattacharjee A. The Oxford Handbook Neuronal Ion Channels. University Press, Oxford, United Kingdom2019: 1-36Google Scholar, 2Catterall W.A. inherited antiepileptic drugs.Annu. Rev. Pharmacol. Toxicol. 2014; 54: 317-338Crossref PubMed Scopus (106) Google Scholar). However, high-throughput manipulations nucleic acid reagents difficult owing (3Feldman D.H. Lossin C. bench series: Plasmid preparation.MethodsX. 1: 6-11Crossref (4) When transformed into typical Escherichia coli host cells, some cDNAs, particularly NaV1.6, exhibit mutation rate resulting single-nucleotide changes, small deletions, insertion sequence (IS) element integrations. Certain (e.g., JM101, DH5?, DH10B/Top10) appear be more prone while others Stbl2 Epi400 can maintain cDNAs stably, possibly reducing plasmid copy number. even accommodating cell types, do occur render isolated useless experiments. Clones on agar plates early grow large colonies almost invariably corrupt, whereas later-appearing clones have lower incidence approaches been instabilities use aforementioned strains, low (30 °C) growth temperature, selecting only colonies, not allowing liquid cultures reach saturation phase Although precautions increase odds obtaining unaltered DNA, unwanted still emerge. Codon optimization has reported alleviate (4Bertelli S. Barbieri R. Pusch M. Gavazzo P. Gain function sporadic/familial hemiplegic migraine-causing SCN1A mutations: Use optimized cDNA.Cephalalgia. 2019; 39: 477-488Crossref (10) 5Patel R.R. Barbosa Brustovetsky T. N. Cummins T.R. Aberrant epilepsy-associated mutant activity targeted cannabidiol.Brain. 2016; 139: 2164-2181Crossref (65) 6Mason E.R. Wu F. Patel Xiao Y. Cannon S.C. Resurgent gating pore currents induced de novo SCN2A epilepsy mutations.eNeuro. 6 (ENEURO.0141-19.2019)Crossref (9) approach may always desirable because altering rare codons protein transmembrane domains impair translational pausing affect proper folding (7Komar A.A. pause thought co-translational pathway.Trends Biochem. Sci. 2009; 34: 16-24Abstract Full Text PDF (237) 8Mauro V.P. Chappell S.A. critical analysis codon therapeutics.Trends Mol. Med. 20: 604-613Abstract (95) 9Shabalina Spiridonov N.A. Kashina Sounds silence: Synonymous nucleotides key biological regulation complexity.Nucleic Acids Res. 2013; 41: 2073-2094Crossref (151) Furthermore, effective stabilizing does instabilities. Because importance research cost time resources incurred instability, sought determine underlying molecular causes enable rationally designed solution generate few no alterations. cDNA (NCBI accession NM_001165963) stability individual cloned subfragments (Fig. 1A). divided four overlapping cassettes (cassette-1: [c.] 1–1560; cassette-2: c.1555–3218; cassette-3: c.3213–5601; cassette-4: c.5341–6030), cassette-3 exhibited evidenced proportion DNAs having altered restriction fragment patterns. To pinpoint responsible was seven analysis. Two (subfragments 5 7) suggesting they harbored unstable 1B). were serial polymerase chain reaction (PCR) amplification 7 introduce would result events informative about location elements. from fast-growing (appearing after overnight growth) screened digestion identify correct patterns, reasoning those mutations. three (one subfragment 5, informative. had (c.4507G>T c.4594G>T, numbering based open frame [ORF]) introduced in-frame premature stop (E1503X G1532X), third clone single (c.4157A>G) nonsynonymous change (D1386E; Fig. 2A). This raised possibility stabilization occurred secondary either truncation disruption inappropriately expressed portion Analysis surrounding revealed element. Specifically, disrupted putative -35 box predicted Sigma 70 factor 2B). These suggested first truncated translated mRNA transcribed clone. test hypothesis, sets made disrupt (c.4155T>C), -10 (c.4179T>C c.4182T>C), both All clones. whether cassette correlated methionine residues (Met1435, Met1438, Met1459, Met1500) original between earliest PCR-induced mutated ATG ATA. Only ATG>ATA (c.4500G>A; Met1500Ile) cassette-3, indicating serve aberrant translation start 3A). In support idea, high-scoring ribosome-binding site (Shine-Dalgarno sequence; TTTGGAGGT) 13–21 5? (c.4486–4497; 3B). cryptic aligns last six-transmembrane domain (domain IV) encoded protein. Introduction did stabilize entire plasmid, suspected existence elsewhere sequence. While 1 2 individually, assembled together. Using similar strategy led identification tested six cassette-1/2 2029 bp segment (c.749–2778). Mutating (Met-350; c.1048–1050) ATA cassette-1/2. An search additional promoters (see Experimental procedures) positions c.27 c.3113 (Table S1). Cassettes remade gene synthesis promoter S2), then recombined cassette-4. final reconstructed S1) included 60 synonymous stably (Top10 cells) grown at 37 °C. inferred located position 875–903 driving initiated Met-350. Figure 4 illustrates promoters, sites, peptides topographical map channel. Guided initial chose simpler required fewer alterations (NaV1.2, NCBI NM_021007; NM_014191) short frames. native exon–exon junctions close analogous found regions where previously but inserted published ?-globin/IgG chimeric intron intervening (IVS) (GenBank U47120) immediately c.2946 disable c.879G>A (c.897T>C, c.900T>C, c.903C>T) well (c.4155T>C) described earlier. Stabilization NaV1.2 inserting IVS c.4551, substitutions required. For cDNA, c.4281 second c.2370. derived HSPA5 NM_005347) modified so it closely consensus eukaryote splice acceptor sites (designated v2; S3). also examined effects (c.1383T>C) out-of-frame mini-ORF spanning c.1382–1390 (ATG CCA TAG) appeared contribute toxicity (slower rate) partial-length fragment. impact yields therefore construct. (c.4208A>G, c.4211T>C) discovered modifications (TOP10 strain) under usual conditions S4). complete presented Figures S5–S7. Finally, compared performing whole-cell voltage clamp recordings transiently transfected HEK-293T cells. differences overall kinetics activation inactivation current any isoform 5). Additionally, measured dependence inactivation, recovery frequency-dependent rundown 50 Hz without 1, S8). concluded channels.Table 1Biophysical channelsPropertyOriginalnStabilizednHuman NaV1.1V1/2 (mV)?22.4 ± 0.914?23.3 1.714Slope (k)6.5 0.1146.4 0.214V1/2 (mV)?56.9 1.114?58.6 1.514Slope (k)?4.9 0.214?4.5 0.114Recovery tau fast (ms)1.5 0.1121.5 0.112% Tau fast77.5 3.01277.5 3.012Recovery slow (ms)243.9 14.112259.1 14.912Inactivation 0 mV (ms)0.40 0.03140.37 0.0314Frequency-dependent (P300/P1)0.79 0.04120.81 0.0414Human NaV1.2V1/2 (mV)?24.4 1.514?25.3 1.114Slope (k)6.7 0.2146.1 (mV)?68.0 1.514?66.8 (k)?6.0 0.414?5.9 (ms)1.7 0.1121.6 0.115% fast81.2 2.31283.3 2.315Recovery (ms)263.9 38.612264.7 27.815Inactivation (ms)0.42 0.03120.39 0.0214Frequency-dependent (P300/P1)0.70 0.04130.72 0.0214Human NaV1.6V1/2 (mV)?24.0 0.715?25.5 1.116Slope (k)6.0 0.2155.7 0.416V1/2 (mV)?66.5 1.615?66.4 0.816Slope (k)?5.9 0.315?5.9 0.316Recovery (ms)1.4 0.1131.5 0.114% fast81.7 4.11381.7 4.114Recovery (ms)240.5 29.313256.0 29.714Inactivation (ms)0.51 0.02150.52 0.0315Frequency-dependent (P300/P1)0.82 0.05130.82 0.0315 Open table new tab essential disease-causing pharmacological studies discovery (10George Inherited channels.J. Clin. Invest. 2005; 115: 1990-1999Crossref (269) Unfortunately, hindered intrinsic bacteria, unclear origin until our study. deduced presence encode apparently speculate coupled negative fitness selection against carrying intact clones, favorable interrupted According model, inactivated. viral documented. cloning hormone encountered difficulties propagating inherent molecule. traced could neutralized TATA (11Bieth E. Cahoreau Cholin Molinas Cerutti Rochiccioli Devauchelle G. Tauber Human receptor: Cloning expression promoter.Gene. 1997; 194: 97-105Crossref (7) liability certain overcome (12Johansen I.E. Intron facilitates virus reestablished vivo.Proc. Natl. Acad. U. 1996; 93: 12400-12405Crossref (75) 13Pu S.Y. R.H. Yang C.C. Jao T.M. Tsai M.H. Wang J.C. Lin H.M. Chao Y.S. Yueh Successful flavivirus infectious novel method reduce genomes.J. Virol. 2011; 85: 2927-2941Crossref (70) appreciated should considered troubleshooting low-yield plasmids. exact manner fragments exert unknown. integration products, contain domains, plasma membrane leading due stress. It possible product forms inclusion bodies. potentially (c. 875–903), Small ORFs promote accumulation peptidyl-tRNA inhibition bacteriophage lambda two-codon barl minigene (14Cruz-Vera L.R. Hernandez-Ramon Perez-Zamorano B. Guarneros dissociation ribosome during depends nature decoding interaction.J. Biol. Chem. 2003; 278: 26065-26070Abstract (29) 15Hernández-Sánchez J. Valadez J.G. Herrera J.V. Ontiveros Lambda bar minigene-mediated involves starvation tRNA.EMBO 1998; 17: 3758-3765Crossref (45) Previous successfully include specialize restricted Scholar) strategies Bertelli et al. modify incorporated mirrored NaV1.5, Inspection strategic inadvertently specific (c.900T>C, c.903T>C) (c.4158C>T) (c.4182T>C) 3, Shine–Dalgarno 3 (c.4482A>G, c.4485T>G). likely reasons plasmid. (5Patel (6Mason specifically. Despite success natural usage whenever Rare enables cotranslational nascent polypeptide. Membrane proteins ion vulnerable effects. example, ether-a-go-go-related (hERG) potassium alters its biosynthesis heterologous significant difference (16Bertalovitz A.C. Badhey M.L.O. McDonald T.V. modification KCNH2 affects characteristics hERG channel.J. 2018; 293: 12120-12136Abstract (6) widespread confound efforts disease-associated variants. alleviates concern, specifically addresses mechanism preserves assessed primarily small-volume cultures. Deviations patterns qualitative index showing semiquantify degree digested h °C NcoI (New England BioLabs) unless otherwise specified volume 20 ?l before evaluating 1% agarose gel electrophoresis. Overnight Top10 culture surrogate plasmid-related toxicity, poor yields. digest pattern subjected Sanger sequencing commercial vendor (Eurofins Genomics). Overlapping amplified PCRs Q5 Hot Start High Fidelity 2x Master mix Biolabs) primer pairs listed Table S1. Cycling were: 98 min; followed 35 cycles 30 s, 55 10 72 45 s; min. Fragments purified NucleoBond Gel/PCR purification system ligated pCR4Blunt-TOPO (Invitrogen/ThermoFisher Scientific). Three microliter ligation reactions transform chemically competent One Shot (Invitrogen) plated LB supplemented kanamycin (50 ?g/ml). Large kanamycin-resistant ml medium miniprep spin columns EcoRI Biolabs). wild-type sequenced directions M13F M13R primers potential Berkeley Neural Network Promoter Prediction tool (http://www.fruitfly.org/seq_tools/promoter.html) sense strand 0.9 cutoff score. searched BPROM threshold 0.2 (17Solovyev V. Salamov Automatic annotation microbial genomes metagenomic sequences.in: Li R.W. Metagenomics Applications Agriculture, Biomedicine Environmental Studies. Nova Science Publishers, Hauppauge, NY2011: 61-78Google Element Search Tool pLOT Mapper 0.75 (http://www.plasmidplotter.com/). Potential ORFs, ORF, Mapper's Shine Dalgarno/ORF finder minimal ORF sizes set greater than 200 amino acids 0.8 mini similarly specifying maximal size five acids. Primers mutation(s) interest, overlap bp, melting temperature (Tm) custom software. PCR performed ng template High-Fidelity 2X Mix 25 according manufacturer's recommendations except extension 80 s per kb total cycles. DpnI min °C, 2.5 standard heat shock protocol selected ?g/ml kanamycin. ranking Sigma70 3213 (bp 1–1560) 1555–3213) synthesized double-stranded (gBlocks, Integrated Technologies) top pCR4-Blunt (Invitrogen). Bacterial analyzed sequencing. pCR4 vector subsequently subcloned below). constructs entirety beginning through polyadenylation signal. homology arms respective targets. linearized Gibson Assembly NEBuilder HiFi BioLabs). Introns (exon 15/16 junction) c.4551 25/26 NaV1.2. c.875–903. c.2370, exons 14 15, c.4370, 22 23. (c.4119A>G, c.4122T>C) promoter. Final pIR-CMV (NaV1.1, NaV1.2) (18Kahlig K.M. Saridey S.K. Kaja Daniels M.A. Wilson Multiplexed transposon-mediated transfer cells.Proc. 2010; 107: 1343-1348Crossref (59) pcDNA4/TO (NaV1.6; ThermoFisher). Each construct internal entry (IRES2) fluorescent mScarlet. Heterologous HEK293T (CRL-3216, American Type Culture Collection). Cells 5% CO2 Dulbecco Eagle's 10% fetal bovine serum, mM L-glutamine, units/ml penicillin, streptomycin. cotransfected intron-stabilized ?1 ?2 subunits (2 ?g ratio 10:1:1 NaV1.X:?1:?2 subunits) Qiagen SuperFect reagent (Qiagen). CD8 receptor (CD8-IRES-h?1) enhanced green (EGFP) (EFGP-IRES-h?2), respectively, transfection markers, (19Lossin D.W. Rhodes T.H. Vanoye C.G. Molecular basis epilepsy.Neuron. 2002; 877-884Abstract (287) Whole-cell recording (20Thompson Kahlig variants divergent sensitivity drugs.Epilepsia. 52: 1000-1009Crossref (51) 21Thompson Porter Nontruncating severe myoclonic infancy surface expression.J. 2012; 287: 42001-42008Abstract (26) room Axopatch 200B amplifier (Molecular Devices). Patch pipettes pulled borosilicate glass capillaries (Harvard Apparatus Ltd) multistage P-1000 Flaming-Brown micropipette puller (Sutter Instruments Co) fire-polished microforge (Narashige MF-830) resistance 1.0–2.0 M?. pipette consisted (in mM): NaF, 105 CsF, CsCl, EGTA, HEPES pH adjusted 7.35 CsOH osmolality 300 mOsmol/kg sucrose. continuously perfused bath containing 145 NaCl, KCl, 1.8 CaCl2, MgCl2, glucose 7.35, 310 mOsmol/kg. Ag/AgCl pellet served reference electrode. chemicals purchased Sigma-Aldrich. Voltage pulse generation data collection done Clampex 10.4 capacitance determined integrating capacitive transients step ?120 ?110 filtered 100 kHz low-pass Bessel filtering. Series compensated prediction >70% correction >90% assure command reached microseconds error <3 mV. Leak subtracted online P/4 procedure. filtering digitized kHz. experiments conducted (20–25 °C). Data Clampfit Devices), Microsoft Excel 2019, GraphPad Prism 8.4 (GraphPad Software, Inc). Peak normalized plotted peak density–voltage relationship. elicited 500 ms pulses ranging –120 mV, tail holding conductance (GNa) calculated GNa = I/(V – Erev), I current, V potential, Erev reversal linear regression I-V curve each cell. maximum ?80 40 calculate (protocol figure insets), fitted Boltzmann G/Gmax= (1 + exp[(V V1/2)/k])?1, V1/2 indicates half-maximal k slope describing steady-state plotting postpulse prepulse increments. plot function. Time-dependent evaluated two-step protocol. –10 inactivate channels. varying periods 3000 ms. fitting two-exponential Frequency dependent (4 ms) frequency Hz. Results mean SEM. Statistical comparisons unpaired two-tailed Student's t-test one-way ANOVA Tukey post-test. p < 0.05 statistically significant. vectors study available Addgene repository (accession numbers: - 162278; 162279; 16280). article. L. serves Scientific Advisory Board Amgen, Inc received grant Praxis Precision Medicines, Merck & Co unrelated work. authors thank Linnenbank providing software image 4). J.-M. D. H. all data. contributed writing