Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.
Bottom Line: Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Na(v)1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Na(v)1.7-DeltaR1370-L1374).Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type.In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Na(v)1.7.
Affiliation: Department of Medical Genetics, University of Cambridge, UK.Show MeSH
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Mentions: We therefore switched to analysis of the FLAG-tagged SCN9A constructs in undifferentiated PC12 cells. Using the monoclonal anti-Nav1.7 antibody we could see an obvious intracellular staining in cells transfected with the wild-type construct (Figure 2a and Supp. Figure S6). Furthermore, in a proportion of cells we could see a faint but distinct ‘rim’ of staining which co-localized with the plasma membrane marker pan cadherin (Figure 2a). In contrast, cells transfected with the mutant channels Nav1.7-ΔR1370-L1374 and Nav1.7-R896Q typically showed no plasma membrane staining for Nav1.7, although a similar number of cells as in the wild-type transfected cells showed strong intracellular staining. This suggests that the mutations caused abnormal trafficking of the channels to the plasma membrane compared to WT (Figure 2a and Supp. Figure S6). However, this phenotype was not the same in every cell as a minority of mutant-transfected cells appeared to show some Nav1.7 staining at the plasma membrane. To quantify this result we counted at least 300 transfected cells over 3 coverslips for each construct (Figure 2b). This showed that there were significantly more WT-transfected cells with Nav1.7 plasma membrane staining than the number of mutant-transfected cells with Nav1.7 plasma membrane staining. This data suggests that both the missense and in-frame deletion pore mutations hamper the surface expression of Nav1.7, and may explain the reason for the loss of pain phenotype seen in these patients.
Affiliation: Department of Medical Genetics, University of Cambridge, UK.