Transcript No Slide Title

Do Sodium Channel α-α Interactions Contribute to Loss-of-Function
Observed in Brugada Syndrome?
Krekwit
Xi
2,3
Du,
• Brugada syndrome (BrS) is an inherited cardiac
disease with an autosomal dominant pattern which
however also displays incomplete penetrance.
Eckhard
2Heart
2
Ficker,
and Isabelle
1,2,3
Deschênes
3Department
METHODS
• 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)
• 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:
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
2
Liu,
of Physiology & Biophysics,
& Vascular Research Center, and
of Biomedical Engineering,
MetroHealth Campus of Case Western Reserve University, Cleveland, Ohio
INTRODUCTION
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
Haiyan
RESULTS
WT (0.3 µg) WT (0.15 µg)WT+L325R L325R
-200
*
-400
*
-600
-800
-1000
Figure 1: Diagram of hNav1.5. Circles represent the mutations
and/or polymorphisms that are characterized in this study.
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.
Tetramer
Trimer
Dimer
• 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)
SUMMARY
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.
0
Current Density @ -20 mV (pA/pF)
1Department
1,2
Shinlapawittayatorn,
SCN5A-WT (n=12)
SCN5A-L567Q (n=9)
• 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 a-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.
CONCLUSIONS
• 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.
SCN5A-H558R + SCN5A-L567Q (n=14)
SCN5A-WT + SCN5A-L567Q (n=9)
WT : L325 cDNA ratios
A
+60 mV
B
• Whole-cell sodium currents were measured at room
temperature using the patch clamp technique in the
whole-cell configuration.
-120 mV
• 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.
*
• Furthermore, binomial analysis was used to
determine subunit composition in a channel complex.
C
• Theoretically, since the biophysical properties of the mutated
channels were also not significantly altered, a BrS
phenotype would not be expected.
-80 mV
• Our experiments using BrS mutations, now suggest the idea
of a dimerization of sodium channel α-subunits.
D
ACKNOWLEDGEMENT
3. Disease-causing sodium channel mutations
have been shown to have a dominant negative
effect on wild type channels.4
This work was supported by an AHA Pre-Doctoral Fellowship
from the Great Rivers Affiliate (KS) and an AHA Scientist
Development Grant (ID).
-30 mV
-65 mV
-120
mV
HYPOTHESIS
Therefore, we hypothesized that the dominantnegative effect seen in some Brugada Syndrome
mutations is due to interactions between sodium
channel a-subunits.
• The BrS mutation SCN5A-L325R produced, as previously
described4, a dominant-negative effect on WT suggesting a
possible interaction between sodium channels a-subunits.
-120 mV
-140
mV
Table 1: Prediction of the response in channels expressed in
HEK-293 cells transfected with a 1:1 mixture of WT (○) and
mutant (●) channels.
-30 mV
Interpulse Interval
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
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