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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
                                                                                                                                                                                                                                        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
                                                                                                                                                                                                                                        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
  multimers.                                                                                                                                                                                                                            normalized mean current densities (■)
                                                                                                                                                                                                                                        measured in cells transfected with different
                                                                 • 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
  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

               HYPOTHESIS                                                                                                                                                            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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