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