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Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

Cox JJ, Sheynin J, Shorer Z, Reimann F, Nicholas AK, Zubovic L, Baralle M, Wraige E, Manor E, Levy J, Woods CG, Parvari R - Hum. Mutat. (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Cambridge, UK.

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Electrophysiological characterisation of HEK293 cells transiently transfected with wild-type and mutant SCN9A. (A) Phase contrast, EGFP and DsRed2 fluorescence of HEK293 cells transiently co-transfected with plasmids expressing SCN9A+DsRed2 and SCN1B+SCN2B+EGFP under a CMV promoter. Note that in the phase contrast with the patch pipette one of the fluorescently positive cells is missing as it was used in the previous patch experiment. (B) Current responses to 50 ms voltage steps in 5 mV increments between −110 and +60 mV from a holding potential of −100 mV, in a whole cell voltage clamp recording applied at ∼0.5 Hz for cells co-expressing either WT or mutant Nav1.7 (as indicated) with the β-subunits, identified by their fluorescence as shown in (A). The inset shows the voltage pulse protocol. (C) Current-voltage relationship of peak currents as shown in (B) normalised for cell size (pA/pF). ▪ Nav1.7+Navβ1+Navβ2 (n=5),  Nav1.7-ΔR1370-L1374+Navβ1+Navβ2 (n=5),  Nav1.7-R896Q+NaVβ1+Navβ2 (n=7),  Navβ1+Navβ2 only (n=5). WT data was fitted with a Boltzmann equation y = (A2+(A1-A2)/(1+exp((V0.5-x)/k)(x-Vrev), where V0.5 = −33 mV, k= 2 mV Vrev= 65 mV.
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fig03: Electrophysiological characterisation of HEK293 cells transiently transfected with wild-type and mutant SCN9A. (A) Phase contrast, EGFP and DsRed2 fluorescence of HEK293 cells transiently co-transfected with plasmids expressing SCN9A+DsRed2 and SCN1B+SCN2B+EGFP under a CMV promoter. Note that in the phase contrast with the patch pipette one of the fluorescently positive cells is missing as it was used in the previous patch experiment. (B) Current responses to 50 ms voltage steps in 5 mV increments between −110 and +60 mV from a holding potential of −100 mV, in a whole cell voltage clamp recording applied at ∼0.5 Hz for cells co-expressing either WT or mutant Nav1.7 (as indicated) with the β-subunits, identified by their fluorescence as shown in (A). The inset shows the voltage pulse protocol. (C) Current-voltage relationship of peak currents as shown in (B) normalised for cell size (pA/pF). ▪ Nav1.7+Navβ1+Navβ2 (n=5), Nav1.7-ΔR1370-L1374+Navβ1+Navβ2 (n=5), Nav1.7-R896Q+NaVβ1+Navβ2 (n=7), Navβ1+Navβ2 only (n=5). WT data was fitted with a Boltzmann equation y = (A2+(A1-A2)/(1+exp((V0.5-x)/k)(x-Vrev), where V0.5 = −33 mV, k= 2 mV Vrev= 65 mV.

Mentions: Given that plasma membrane staining was occasionally seen when the mutant channels were overexpressed, we decided to investigate how the mutations affected the biophysical properties of the sodium channel. To do this, plasmids bearing either WT SCN9A or the mutant sequences were co-expressed in HEK293A cells with the auxiliary sodium channel β1 and β2 subunits (encoded by SCN1B and SCN2B). Whole cell voltage clamp recordings from cells co-expressing wild-type Nav1.7 with the β1β2 subunits, revealed a voltage-gated Na+ current with a peak amplitude of −685 ± 134 pA/pF at −20 mV (n=5), compared with a background current of −13 ± 2 pA/pF (n=5) in cells transfected with the β1 β2 construct alone (p=0.001 for wild-type Nav 1.7 vs control) (Figure 3). The voltage dependence of activation of Nav1.7 could be described by a Boltzmann function, with half maximum activation (V0.5) of −33± 2 mV, k = 2 ± 1 mV and a reversal potential (Vrev) of +65 ± 4 mV (n=5). Voltage dependent inactivation could also be described by a Boltzmann function with half maximal inactivation at −74 ± 2 mV and k = 4.3 ± 0.6 mV (n=5) (Supp. Figure S5). These properties are similar to those described previously for Nav1.7 current [Klugbauer et al., 1995; Cummins et al, 2004; Herzog et al., 2003]. In contrast, cells co-transfected with either of the mutated Nav1.7 subunits plus β1β1, exhibited currents that were not significantly different from those recorded from control cells (Figure 3). The mean peak currents at −20 mV were: −11±3 pA/pF (n=7, p>0.6 vs control) and −13 ± 5 pA/pF (n=5, p>0.9 vs control) for Nav1.7-R896Q and Nav1.7-ΔR1370-L1374 respectively. Hence, the two mutations completely abolish the function of the voltage-gated sodium channel, which is consistent with the complete insensitivity to pain phenotype seen in affected individuals from these two families.


Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

Cox JJ, Sheynin J, Shorer Z, Reimann F, Nicholas AK, Zubovic L, Baralle M, Wraige E, Manor E, Levy J, Woods CG, Parvari R - Hum. Mutat. (2010)

Electrophysiological characterisation of HEK293 cells transiently transfected with wild-type and mutant SCN9A. (A) Phase contrast, EGFP and DsRed2 fluorescence of HEK293 cells transiently co-transfected with plasmids expressing SCN9A+DsRed2 and SCN1B+SCN2B+EGFP under a CMV promoter. Note that in the phase contrast with the patch pipette one of the fluorescently positive cells is missing as it was used in the previous patch experiment. (B) Current responses to 50 ms voltage steps in 5 mV increments between −110 and +60 mV from a holding potential of −100 mV, in a whole cell voltage clamp recording applied at ∼0.5 Hz for cells co-expressing either WT or mutant Nav1.7 (as indicated) with the β-subunits, identified by their fluorescence as shown in (A). The inset shows the voltage pulse protocol. (C) Current-voltage relationship of peak currents as shown in (B) normalised for cell size (pA/pF). ▪ Nav1.7+Navβ1+Navβ2 (n=5),  Nav1.7-ΔR1370-L1374+Navβ1+Navβ2 (n=5),  Nav1.7-R896Q+NaVβ1+Navβ2 (n=7),  Navβ1+Navβ2 only (n=5). WT data was fitted with a Boltzmann equation y = (A2+(A1-A2)/(1+exp((V0.5-x)/k)(x-Vrev), where V0.5 = −33 mV, k= 2 mV Vrev= 65 mV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: Electrophysiological characterisation of HEK293 cells transiently transfected with wild-type and mutant SCN9A. (A) Phase contrast, EGFP and DsRed2 fluorescence of HEK293 cells transiently co-transfected with plasmids expressing SCN9A+DsRed2 and SCN1B+SCN2B+EGFP under a CMV promoter. Note that in the phase contrast with the patch pipette one of the fluorescently positive cells is missing as it was used in the previous patch experiment. (B) Current responses to 50 ms voltage steps in 5 mV increments between −110 and +60 mV from a holding potential of −100 mV, in a whole cell voltage clamp recording applied at ∼0.5 Hz for cells co-expressing either WT or mutant Nav1.7 (as indicated) with the β-subunits, identified by their fluorescence as shown in (A). The inset shows the voltage pulse protocol. (C) Current-voltage relationship of peak currents as shown in (B) normalised for cell size (pA/pF). ▪ Nav1.7+Navβ1+Navβ2 (n=5), Nav1.7-ΔR1370-L1374+Navβ1+Navβ2 (n=5), Nav1.7-R896Q+NaVβ1+Navβ2 (n=7), Navβ1+Navβ2 only (n=5). WT data was fitted with a Boltzmann equation y = (A2+(A1-A2)/(1+exp((V0.5-x)/k)(x-Vrev), where V0.5 = −33 mV, k= 2 mV Vrev= 65 mV.
Mentions: Given that plasma membrane staining was occasionally seen when the mutant channels were overexpressed, we decided to investigate how the mutations affected the biophysical properties of the sodium channel. To do this, plasmids bearing either WT SCN9A or the mutant sequences were co-expressed in HEK293A cells with the auxiliary sodium channel β1 and β2 subunits (encoded by SCN1B and SCN2B). Whole cell voltage clamp recordings from cells co-expressing wild-type Nav1.7 with the β1β2 subunits, revealed a voltage-gated Na+ current with a peak amplitude of −685 ± 134 pA/pF at −20 mV (n=5), compared with a background current of −13 ± 2 pA/pF (n=5) in cells transfected with the β1 β2 construct alone (p=0.001 for wild-type Nav 1.7 vs control) (Figure 3). The voltage dependence of activation of Nav1.7 could be described by a Boltzmann function, with half maximum activation (V0.5) of −33± 2 mV, k = 2 ± 1 mV and a reversal potential (Vrev) of +65 ± 4 mV (n=5). Voltage dependent inactivation could also be described by a Boltzmann function with half maximal inactivation at −74 ± 2 mV and k = 4.3 ± 0.6 mV (n=5) (Supp. Figure S5). These properties are similar to those described previously for Nav1.7 current [Klugbauer et al., 1995; Cummins et al, 2004; Herzog et al., 2003]. In contrast, cells co-transfected with either of the mutated Nav1.7 subunits plus β1β1, exhibited currents that were not significantly different from those recorded from control cells (Figure 3). The mean peak currents at −20 mV were: −11±3 pA/pF (n=7, p>0.6 vs control) and −13 ± 5 pA/pF (n=5, p>0.9 vs control) for Nav1.7-R896Q and Nav1.7-ΔR1370-L1374 respectively. Hence, the two mutations completely abolish the function of the voltage-gated sodium channel, which is consistent with the complete insensitivity to pain phenotype seen in affected individuals from these two families.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Cambridge, UK.

Show MeSH
Related in: MedlinePlus