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Supplementary Methods
Molecular genetics
RNA extraction: For each RNA extraction nerve tissue was homogenized in a micro
tissue grinder containing 800l Trizol reagent (Invitrogen) and 200 g linear
acrylamide (Ambion). The genomic DNA was sheared by passing it through a 26-gauge
needle connected to a 1 ml syringe and transferred to a 1.6 ml microcentrifuge tube.
Chloroform (160l) was added and the sample was vortexed for 30 sec, then
centrifuged to separate the aqueous and organic phases (10,000 x g). Any potentially
contaminating DNA was removed from the RNA by a 1h incubation in DNase 1
(Molecular Probes) at 37 oC followed by 10 min at 75 oC. RNA was precipitated with
isopropanol at –20oC for 1 h prior to pelleting. The pellet was washed with 70%
ethanol, air dried and resolubilized in 30l RNAsecure® (Ambion), heated to 60oC for
10 min, then stored at –80oC. DNA was isolated from the organic phase following
manufacturer’s instructions.
Isolation of cDNA and Na+ channel specific fragments: Degenerate PCR primers were
designed based on aligned Na+ channel amino acid sequences of the turbellarian
flatworm Bdelloura candida1, sea hare Aplysia californica2 and Pacific market squid
Loligo opalescens3 spanning the functional domains of Na+ channels. The first fragment
of the M. arenaria Na+ channel was generated by PCR amplification using degenerate
primers corresponding to S6 domain III (degenerate forward primer 5’GTNATHATIGAYAAYTTYAA-3’) and S6 domain IV (degenerate reverse primer 5’DATIACNGCIATRTACATRTT-3’). This fragment was used as a template for nested
PCR using primers SNC-2 (ATCTGATCACGTGCAATTATTTAATGGCAAGTTCC)
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and SNC-4 (GTGGATCCTCAATGGAACATCCATTCAGC) from L. opalescens3
resulting in a 352-bp fragment. cDNA sequences from that initial M. arenaria Na+
channel fragment were then used to develop 3’(reverse) M. arenaria-specific PCR
primers that were used in conjunction with degenerate forward primers. In this way the
Na+ channel gene sequence was obtained by walking toward the 5’ end from domain
IV. The PCR reactions were carried out in 50l volumes with PfuUltraTM hotstart PCR
master mix (Stratagene) in a PTC-100 thermal cycler (MJ Research) with the following
program: 94oC for 5 min; 35 cycles of (94oC for 30 sec, 50oC for 30 sec and 72oC for 1
min); ending with an extension at 74oC for 10 min. DNA from PCR reactions was
immediately isolated by separation on a 1.8% low-melt agarose gel (NuSieve GTG,
FMC Biopolymers) in 1x TAE buffer (40mM Tris-acetate, 1mM EDTA) with ethidium
bromide/UV visualization, and the DNA was excised. The isolated PCR products were
ligated into pCR4-TOPO® (Invitrogen), transformed into chemically competent E. coli
OneShot® TOP 10 cells (Invitrogen) and selected on LB ampicilin agar plates by
standard methods4. Plasmid DNA was isolated from a single colony with a Qiagen
plasmid isolation kit (Qiagen) following the manufacturer’s instructions and sequenced.
Alignment of DNA sequences was accomplished using Sequencher software
(GeneCodes Corp.). The predicted amino acid sequence of M. arenaria Na+ channel subunits was aligned to those of both vertebrate and invertebrate using Clustal W5.
Determination of Na+ channel sequence from individuals: genomic DNA and cDNA
sequence was obtained for each of the four pore regions using M. arenaria-specific PCR
primers (domain I -5’ CTT TGA CAA CTT TGG CTG GG 3’ and 5’ AAG GAA CCC
AGC AGA ATG AC 3’; domain II- 5’ TCC GGG GAT GAT ATT CCA AG 3’ and 5’
CCA CCA GAT TTC CTA TAA AG 3’; domain III- 5’ ATT ATT CTT GGG AAA
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ATT CT 3’ and 5’ GTA AAT ATA CAT CAA CGG GT 3’; domain IV- 5’ CGG TGG
ACT AGA CGA TGT TT 3’ and 5’ TAA AGG GTA ACT GCC ACA CG 3’). PCR
reactions were carried out as detailed above.
Construction of Na+ channel mutations
Materials for the construction of the pCDM8 Nav 1.2a E945D were supplied as follows:
all restriction enzymes, Antarctic phosphatase and diluents from New England
BioLabs); bacterial cells both Top 10/P3 and DH5 (Invitrogen); PfuUltraTM hotstart
PCR master mix (Stratagene); FastLink ligation kit (Epicentre); QIAquick plasmid
purification kits (Qiagen); agarose (NuSieve GTG, FMC Biopolymers); PCR primers
(Integrated DNA Technologies, Inc); antibiotics and general chemicals (Sigma). All
reagents were used according to protocols provided by the manufacturer.
Plasmid pCDM8 Nav 1.2a6 contains the full-length rIIa Na+ channel gene. The
shuttle vector plasmid pBluescript SK+/rNav 1.2a (Xma I> Bst EII) contains the Xma I
(nucleotide 1896 in the loop between Domains I and II) to Bst EII (nucleotide 4636 in
domain IV region S1) fragment of full-length rat pCDM8/ Nav 1.2a (rIIa).
Briefly, the E945D mutant was prepared in pBluescript SK+/rNav 1.2a (Xma I> Bst
EII) by a two-step protocol (detailed below) using 2 mutagenic primers and 2 restriction
site primers. Following mutagenesis, a Sph I>Blp I fragment was ligated into full-length
pCDM8 Nav 1.2a. The sequence of the final construct was confirmed by restriction
mapping and DNA sequence analysis.
Step 1 of the two-step mutagenesis protocol used primers to make each arm by
amplifying DNA from pCDM8/Nav 1.2a containing rIIa. The primers for left and right
arms are: left arm upstream the restriction site Sph I to the mutation (Forward) 5’ CTG
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ACC CTG GTG GGC ATC ATC GTC TTC ATT TT 3’ and (Reverse) 5’ GCA GTC
CCA CAT GGT GTC TAT CCA CTC CCC ACA CA 3’; right arm mutation to
downstream the Bgl II restriction site, (Forward) 5’ TGT GTG GGG AGT GGA TAG
ACA CCATGT GGG ACT GC 3’ and (Reverse) 5’ TTG TCT ATT TCT ATGTC GTG
TGG TGG GAG ATA CA 3’. PCR parameters and amplicon extraction followed
standard protocols. Both arms were mixed as PCR templates and the full-length
fragment was amplified using the forward left arm primer and reverse right arm primer.
The resulting full-length amplicon was digested with restriction endonucleases Shp I
and Bgl II prior to isolation by separation on agarose gel and gel extraction. The shuttle
vector pBluescript SK+/rNav 1.2a (Xma I> Bst EII) was digested with both Shp I and
Bgl II followed by treatment with phosphatase and isolation of the backbone by
separation on agarose gel with gel extraction. DNA concentration was determined. The
full-length fragment and the shuttle vector were ligated and transformed into
DH5bacterial cells following the manufacturers’ recommendations. Positive
transformants were selected on media containing 50 μg ml-1 ampicillin. DNA was
extracted from clones and the plasmid sequence was confirmed by sequencing of the
fragment as described above using the left arm forward and right arm reverse primers.
The plasmid pCDM8 Nav 1.2a was prepared by digestion with Sph I and Blp I followed
by phosphatase treatment and isolation of the backbone fragment through gel extraction.
A Sph I/Blp I fragment was digested from the shuttle vector followed by gel isolation.
The pCDM8 Nav 1.2a backbone and the Sph I/Blp I fragment were ligated and the
constructs were transformed into TOP 10/P bacterial cells. Positive transformats were
selected on media containing 50 μg ml-1 ampicillin and 7.5 μg ml-1 tetracycline. Large
batches of plasmid were isolated using Maxi plasmid kit and DNA concentration
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determined. The sequence of the insert in the new plasmid pCDM8 Nav 1.2a E945D
was confirmed by sequencing the final large batch of isolated plasmid using the left arm
forward primer and 5’ AAC CTT GTC AGC ATA CTC C 3’ (downstream of Blp I
restriction site).
References
1. Jeziorski, M.C., Greenberg, R.M. & Anderson, P.A.V. Cloning of a putative voltagegated sodium channel from the turbellarian flatworm Bdelloura candida. Parasitol.
115, 289-296 (1997).
2. Dyer, J.R., Johnston, W.L., Castellucci, V.F. & Dunn, R.J. Cloning and tissue
distribution of the Aplysia Na+ channel α-subunit cDNA. DNA and Cell Biology 16,
347-356 (1997).
3. Rosenthal, J.J.C. & Gilly, W.F. Amino acid sequence of a putative sodium channel
expressed in the giant axon of the squid Loligo opalescens. Proc. Nat. Acad. Sci.
USA 90, 10026-10030 (1993).
4. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor Press, Cold Spring Harbor, NY, ed 2, 1989).
5. Thompson, J.D., Higgens, D.G., Gibson, T.J. CLUSTAL W: Improving the
sensitivity of progressive multiple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice. Nucleic Acids Research
22, 4673-4680 (1994).
6. Linford, N.J., Cantrell, A.R., Qu, Y., Scheuer, T. & Catterall, W.A. Interaction of
batrachotoxin with the local anesthetic receptor site in transmembrane segment
IVS6 of the voltage-gated sodium channel. Proc Nat. Acad. Sci, USA 95, 1394713952 (1998).