E1674 Sheynin et al. Construction of expression plasmids The wild-type SCN9A polio IRES DsRed2 construct (FLRED) and the SCN1B encephalomyocarditis virus (ECMV) IRES SCN2B polio IRES EGFP construct (JC5) were generated as previously described [Cox et al., 2006]. These bear either SCN9A and DsRed2 (FLRED) or SCN1B, SCN2B and EGFP (JC5) on the same vector expressed from the same promoter and the inclusion of DsRed2 and EGFP in these constructs helped in the identification of positively co-transfected cells when patch clamping. The Nav1.7-R896Q and Nav1.7 - ΔR1370-L1374 mutations were introduced into FLREDN using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) or by recombinant PCR respectively. The FLAG (DYKDDDDK) epitope was introduced into the above WT and mutant constructs into the domain I extracellular S1-S2 linker in frame between Pro 149 and Asp 150, using recombinant PCR techniques. For the immunocytochemistry experiments, the polio IRES-DsRED2 fragment was removed from the WT and mutant constructs so that only the Nav1.7 protein was expressed. All constructs were sequenced to verify the desired mutation and epitope and to ensure the lack of other introduced variations. Immunocytochemistry Rat pheochromocytoma cells (PC12) cultured in DMEM supplemented with 10% horse serum and 5% FCS, were transiently transfected with the WT or either of the 2 mutant constructs using lipofectamine 2000. The JC5 construct was not co-transfected as endogenous beta subunits are present within PC12 cells. One day after transfection, the cells were fixed in ice-cold methanol for 5 mins and then permeabilized using acetone. Fixed cells were blocked with 3% BSA for 40 mins. Staining was carried out using the following antibodies: mouse monoclonal anti-Nav1.7 (1:250; Neuromab), mouse monoclonal anti-FLAG M2 (1:1000; Sigma), and rabbit polyclonal anti-Pan Cadherin (1:100; Sigma); all detected with appropriate Alexa Fluor secondary antibodies (1:250; Invitrogen). Slides were mounted using Prolong Gold antifade reagent with DAPI (Invitrogen) and examined using a Zeiss LSM510 META confocal laser-scanning inverted microscope (Carl Zeiss) equipped with an argon-krypton laser beam. At least 100 consecutive transfected cells per x3 coverslips were assessed for plasma membrane localization for each construct. To prevent bias the quantifying person was blinded against the nature of the analysed construct. Data was statistically analyzed using a Fisher’s two-tailed exact test. Electrophysiology HEK293A cells (QBiogene), cultured in DMEM supplemented with 5% FCS, were transiently transfected with plasmids expressing either WT or mutant Nav1.7 plus DsRed2 and/or SCN1B plus SCN2B plus EGFP using lipofectamine 2000. Whole-cell voltage clamp protocols were performed 2-3 days after transfection as previously described [Cox et al., 2006]. The bath solution contained (in mM): 3 KCl, 140 NaCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 1 glucose (pH7.4 with NaOH) and the patch pipette solution contained (in mM): 107 CsF, 10 NaCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 10 TEACl, 10 EGTA (pH 7.2 with CsOH). RESULTS Novel mutations identified in SCN9A Mapping and DNA sequence analysis in an Israeli Bedouin family Since the patients belong to the population of Bedouins in the south of Israel where congenital insensitivity to pain with anhydrosis (CIPA) is prevalent we firstly excluded linkage to the TrkA gene, assuming homozygosity in which the two alleles are identical by descent, by the use of microsatellite markers adjacent to the gene (data not shown). We next analyzed whether the patients presented homozygosity near the SCN9A gene, which was reported to be mutated in CIP patients [Cox et al., 2006; Goldberg et al., 2007; Ahmad et al., 2007; Nilsen et al., 2009]. Linkage to chromosome 2 (q23.3-q24.3), position 153,102,193–168,960,515 (NCBI Build 36.1 reference sequence) was confirmed by analysis of all family members for microsatellite markers in the region (Figure 1a). We searched for mutations in each of the coding exons and their intronic borders of this gene using DNA of patient ip3 and identified a single homozygous base substitution in coding exon 15 c.2687G>A resulting in amino