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-1000 -800 -600 -400 -200 0 C u rren t D en sity @ -20 m V (p A /pF) WT (0.3 µg) WT (0.15 µg) WT+L325R L325R * * • Using site directed mutagenesis, we introduced the BrS mutations SCN5A- L325R and SCN5A-L567Q, and the SCN5A- H558R polymorphism on the human cardiac sodium channel, hNav1.5 (Fig. 1) • We transiently transfected HEK-293 cells with recombinant: - SCN5A-WT - SCN5A-L325R - SCN5A-WT + SCN5A-L325R (10:1, 1:1, and 4:1) - SCN5A-L567Q - SCN5A-H558R + SCN5A-L567Q (1:1) - SCN5A-WT + SCN5A-L567Q (1:1) • Whole-cell sodium currents were measured at room temperature using the patch clamp technique in the whole-cell configuration. • Furthermore, binomial analysis was used to determine subunit composition in a channel complex. • The BrS mutation SCN5A-L325R produced, as previously described 4 , a dominant-negative effect on WT suggesting a possible interaction between sodium channels - subunits. • Using a binomial distribution, our results indicate that sodium channels can organize as a multi-channel complex with a configuration suggesting an interaction of two -subunits. • The BrS SCN5A-L567Q mutation did not produce significant biophysical changes compared to WT. Biophysical properties remained unchanged when SCN5A-L567Q was co-expressed with either SCN5A-H558R or SCN5A-WT. • Interestingly, when SCN5A-L567Q was co- expressed with either the SCN5A-H558R polymorphism or the SCN5A-WT channels, current densities were greatly reduced. Do Sodium Channel Do Sodium Channel α α - - α α Interactions Contribute to Loss-of-Function Interactions Contribute to Loss-of-Function Observed in Brugada Syndrome? Observed in Brugada Syndrome? Krekwit Shinlapawittayatorn, Krekwit Shinlapawittayatorn, 1,2 1,2 Xi Du, Xi Du, 2,3 2,3 Haiyan Liu, Haiyan Liu, 2 2 Eckhard Ficker, Eckhard Ficker, 2 2 and and Isabelle Isabelle Desch Desch ênes ênes 1,2,3 1,2,3 1 1 Department of Physiology & Biophysics, Department of Physiology & Biophysics, 2 2 Heart & Vascular Research Center, and Heart & Vascular Research Center, and 3 3 Department of Department of Biomedical Engineering Biomedical Engineering , , MetroHealth Campus of Case Western Reserve University, Cleveland, MetroHealth Campus of Case Western Reserve University, Cleveland, Ohio Ohio • Brugada syndrome (BrS) is an inherited cardiac disease with an autosomal dominant pattern which however also displays incomplete penetrance. • The pathogenesis of BrS has mainly been associated with mutations in the cardiac sodium channel gene, SCN5A, which ultimately result in a decrease in sodium currents. • Recently, the L325R mutation has been proposed to cause BrS through a dominant-negative effect. • Dominant-negative effects are usually the consequence of mutant subunits assembling with wild-type (WT) into non-functional channel multimers. • In contrast, sodium channel α-subunits are not believed to oligomerize. • However, increasing bodies of evidence tend to suggest that there is, contrary to traditional beliefs, a sodium channel α-α interaction: 1.Single-channel experiments have demonstrated a tendency for even numbers of channels to occur within a patch. Importantly, no single opening peak was present in an amplitude distribution histogram. 1,2 2.It has previously been shown that a common sodium channel polymorphism located on a separate allele can restore the function of a disease causing mutation. 3 3.Disease-causing sodium channel mutations have been shown to have a dominant negative effect on wild type channels. 4 INTRODUCTION METHODS • While most BrS mutations produce loss-of- function in sodium channels leading to a reduction in current density, the SCN5A-L567Q mutation had no noticeable effect on sodium current density. • Theoretically, since the biophysical properties of the mutated channels were also not significantly altered, a BrS phenotype would not be expected. • However, the reduction in current density observed when the mutation was co-expressed with WT channels could produce the BrS phenotype. • Our results suggest that some BrS mutations, although displaying minimal biophysical alterations, may produce the clinical phenotype through an α-α interaction thus leading to a reduction in sodium current density. • Our experiments using BrS mutations, now suggest the idea of a dimerization of sodium channel α-subunits. CONCLUSIONS REFERENCES Figure 1: Diagram of hNav1.5. Circles represent the mutations and/or polymorphisms that are characterized in this study. RESULTS SUMMARY Figure 4: A. Current densities of WT (0.3 μg), SCN5A-L567Q (0.3 μg), SCN5A-H558R (0.15 μg) + SCN5A-L567Q (0.15 μg), and SCN5A-WT (0.15 μg) + SCN5A-L567Q (0.15 μg). Electrophysiological characterization of: (●) SCN5A-L567Q, (▲) SCN5A-H558R + SCN5A- L567Q, and (▼) SCN5A-WT + SCN5A-L567Q in which currents were compared to (■) SCN5A- WT. B. I/V relationship. C. Steady State Inactivation. D. Recovery from Inactivation. *p<0.05 ACKNOWLEDGEMENT This work was supported by an AHA Pre-Doctoral Fellowship from the Great Rivers Affiliate (KS) and an AHA Scientist Development Grant (ID). 1. Aldrich RW, Corey DP, Stevens CF. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature. 1983;306:436-41. 2.Undrovinas AI, Fleidervish IA, Makielski JC. Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine. Circ Res. 1992;71:1231-41. 3. Poelzing S, Forleo C, Samodell M, Dudash L, Sorrentino S, Anaclerio M, Troccoli R, Iacoviello M, Romito R, Guida P, Chahine M, Pitzalis M, Deschenes I. SCN5A polymorphism restores trafficking of a Brugada syndrome mutation on a separate gene. Circulation. 2006;114:368-76. 4. Keller DI, Rougier JS, Kucera JP, Benammar N, Fressart V, Guicheney P, Madle A, Fromer M, Schlapfer J, Abriel H. Brugada syndrome and fever: genetic and molecular characterization of patients carrying SCN5A mutations. Cardiovasc Res. 2005;67:510-9. Therefore, we hypothesized that the dominant-negative effect seen in some Brugada Syndrome mutations is due to interactions between sodium channel - subunits. HYPOTHESIS Table 1: Prediction of the response in channels expressed in HEK-293 cells transfected with a 1:1 mixture of WT (○) and mutant (●) channels. Figure 2: Current densities of WT (0.3 μg), WT (0.15 μg), WT (0.15 μg) + SCN5A-L325R (0.15 μg), and SCN5A-L325R (0.3 μg). No Sodium currents were present in L325R transfected cells. Interestingly, when WT and L325R channels were co-transfected in 1:1 ratio, the peak current density was reduced to only 29.8±6.2% of the control condition where 100% of WT channels were expressed. This finding suggests that the L325R alleles exerts a dominant negative effect on WT channels. n = 6-10 cells per each group, *p<0.05. Figure 3: Binomial analysis of subunit composition. Binomial analysis was used to determine subunit composition in a channel complex using 10:1, 4:1 and 1:1 WT:L325R ratios. Interestingly, it is shown that normalized mean current densities (■) measured in cells transfected with different WT:L325R cDNA ratios are best fit by the dimer configuration. SCN5A-WT (n=12) SCN5A-L567Q (n=9) SCN5A-H558R + SCN5A-L567Q (n=14) SCN5A-WT + SCN5A-L567Q (n=9) A +60 mV -80 mV -120 mV B -65 mV -140 mV -30 mV -120 mV C -30 mV -120 mV Interpulse Interval D * * Tetramer Trimer Dimer WT : L325 cDNA ratios
description
WT (0.3 µg). WT (0.15 µg). WT+L325R. L325R. *. *. SCN5A -WT (n=12). SCN5A -L567Q (n=9). -30 mV. SCN5A -H558R + SCN5A- L567Q (n=14). -65 mV. SCN5A -WT + SCN5A- L567Q (n=9). Tetramer Trimer Dimer. -120 mV. -140 mV. A. B. *. *. C. D. -30 mV. -120 mV. Interpulse Interval. - PowerPoint PPT Presentation
Transcript of WT (0.3 µg)
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• Using site directed mutagenesis, we introduced the BrS mutations SCN5A-L325R and SCN5A-L567Q, and the SCN5A-H558R polymorphism on the human cardiac sodium channel, hNav1.5 (Fig. 1)
• We transiently transfected HEK-293 cells with recombinant:
- SCN5A-WT- SCN5A-L325R- SCN5A-WT + SCN5A-L325R (10:1, 1:1, and
4:1)- SCN5A-L567Q- SCN5A-H558R + SCN5A-L567Q (1:1)- SCN5A-WT + SCN5A-L567Q (1:1)
• Whole-cell sodium currents were measured at room temperature using the patch clamp technique in the whole-cell configuration.
• Furthermore, binomial analysis was used to determine subunit composition in a channel complex.
• The BrS mutation SCN5A-L325R produced, as previously described4, a dominant-negative effect on WT suggesting a possible interaction between sodium channels -subunits.
• Using a binomial distribution, our results indicate that sodium channels can organize as a multi-channel complex with a configuration suggesting an interaction of two -subunits.
• The BrS SCN5A-L567Q mutation did not produce significant biophysical changes compared to WT. Biophysical properties remained unchanged when SCN5A-L567Q was co-expressed with either SCN5A-H558R or SCN5A-WT.
• Interestingly, when SCN5A-L567Q was co-expressed with either the SCN5A-H558R polymorphism or the SCN5A-WT channels, current densities were greatly reduced.
Do Sodium Channel Do Sodium Channel αα--αα Interactions Contribute to Loss-of-Function Interactions Contribute to Loss-of-FunctionObserved in Brugada Syndrome?Observed in Brugada Syndrome?
Krekwit Shinlapawittayatorn,Krekwit Shinlapawittayatorn,1,21,2 Xi Du,Xi Du,2,32,3 Haiyan Liu, Haiyan Liu,22 Eckhard Ficker, Eckhard Ficker,22 and and Isabelle DeschIsabelle Deschênesênes1,2,31,2,3
11Department of Physiology & Biophysics, Department of Physiology & Biophysics, 22Heart & Vascular Research Center, and Heart & Vascular Research Center, and 33Department of Biomedical EngineeringDepartment of Biomedical Engineering, , MetroHealth Campus of Case Western Reserve University, Cleveland, OhioMetroHealth Campus of Case Western Reserve University, Cleveland, Ohio
• Brugada syndrome (BrS) is an inherited cardiac disease with an autosomal dominant pattern which however also displays incomplete penetrance.
• The pathogenesis of BrS has mainly been associated with mutations in the cardiac sodium channel gene, SCN5A, which ultimately result in a decrease in sodium currents.
• Recently, the L325R mutation has been proposed to cause BrS through a dominant-negative effect.
• Dominant-negative effects are usually the consequence of mutant subunits assembling with wild-type (WT) into non-functional channel multimers.
• In contrast, sodium channel α-subunits are not believed to oligomerize.
• However, increasing bodies of evidence tend to suggest that there is, contrary to traditional beliefs, a sodium channel α-α interaction:
1. Single-channel experiments have demonstrated a tendency for even numbers of channels to occur within a patch. Importantly, no single opening peak was present in an amplitude distribution histogram.1,2
2. It has previously been shown that a common sodium channel polymorphism located on a separate allele can restore the function of a disease causing mutation.3
3. Disease-causing sodium channel mutations have been shown to have a dominant negative effect on wild type channels.4
INTRODUCTION METHODS
• While most BrS mutations produce loss-of-function in sodium channels leading to a reduction in current density, the SCN5A-L567Q mutation had no noticeable effect on sodium current density.
• Theoretically, since the biophysical properties of the mutated channels were also not significantly altered, a BrS phenotype would not be expected.
• However, the reduction in current density observed when the mutation was co-expressed with WT channels could produce the BrS phenotype.
• Our results suggest that some BrS mutations, although displaying minimal biophysical alterations, may produce the clinical phenotype through an α-α interaction thus leading to a reduction in sodium current density.
• Our experiments using BrS mutations, now suggest the idea of a dimerization of sodium channel α-subunits.
CONCLUSIONS
REFERENCES
Figure 1: Diagram of hNav1.5. Circles represent the mutations and/or polymorphisms that are characterized in this study.
RESULTS SUMMARY
Figure 4: A. Current densities of WT (0.3 μg), SCN5A-L567Q (0.3 μg), SCN5A-H558R (0.15 μg) + SCN5A-L567Q (0.15 μg), and SCN5A-WT (0.15 μg) + SCN5A-L567Q (0.15 μg). Electrophysiological characterization of: (●) SCN5A-L567Q, (▲) SCN5A-H558R + SCN5A-L567Q, and (▼) SCN5A-WT + SCN5A-L567Q in which currents were compared to (■) SCN5A-WT. B. I/V relationship. C. Steady State Inactivation. D. Recovery from Inactivation. *p<0.05
ACKNOWLEDGEMENTThis work was supported by an AHA Pre-Doctoral Fellowship from the Great Rivers Affiliate (KS) and an AHA Scientist Development Grant (ID).
1. Aldrich RW, Corey DP, Stevens CF. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature. 1983;306:436-41.2.Undrovinas AI, Fleidervish IA, Makielski JC. Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine. Circ Res. 1992;71:1231-41.3. Poelzing S, Forleo C, Samodell M, Dudash L, Sorrentino S, Anaclerio M, Troccoli R, Iacoviello M, Romito R, Guida P, Chahine M, Pitzalis M, Deschenes I. SCN5A polymorphism restores trafficking of a Brugada syndrome mutation on a separate gene. Circulation. 2006;114:368-76.4. Keller DI, Rougier JS, Kucera JP, Benammar N, Fressart V, Guicheney P, Madle A, Fromer M, Schlapfer J, Abriel H. Brugada syndrome and fever: genetic and molecular characterization of patients carrying SCN5A mutations. Cardiovasc Res. 2005;67:510-9.
Therefore, we hypothesized that the dominant-negative effect seen in some Brugada Syndrome mutations is due to interactions between sodium channel -subunits.
HYPOTHESIS
Table 1: Prediction of the response in channels expressed in HEK-293 cells transfected with a 1:1 mixture of WT (○) and mutant (●) channels.
Figure 2: Current densities of WT (0.3 μg), WT (0.15 μg), WT (0.15 μg) + SCN5A-L325R (0.15 μg), and SCN5A-L325R (0.3 μg). No Sodium currents were present in L325R transfected cells. Interestingly, when WT and L325R channels were co-transfected in 1:1 ratio, the peak current density was reduced to only 29.8±6.2% of the control condition where 100% of WT channels were expressed. This finding suggests that the L325R alleles exerts a dominant negative effect on WT channels. n = 6-10 cells per each group, *p<0.05.
Figure 3: Binomial analysis of subunit composition. Binomial analysis was used to determine subunit composition in a channel complex using 10:1, 4:1 and 1:1 WT:L325R ratios. Interestingly, it is shown that normalized mean current densities (■) measured in cells transfected with different WT:L325R cDNA ratios are best fit by the dimer configuration.
SCN5A-WT (n=12)
SCN5A-L567Q (n=9)
SCN5A-H558R + SCN5A-L567Q (n=14)
SCN5A-WT + SCN5A-L567Q (n=9)
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+60 mV
-80 mV-120 mV
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-65 mV
-140 mV
-30 mV
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-30 mV
-120 mVInterpulse Interval
D
**
TetramerTrimer Dimer
WT : L325 cDNA ratios
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