S. It has been previously shown that mutations in the pore region may affect voltage activation of Nav1.5 and other Nav channels [32?5]. The hypothesis that mutations in the pore region of Nav channels can lead to structural perturbation compromising activation has been put forward [32]. In this context, our immunodetection experiments indicated that the decrease in INa observed in I890T cells could not be explained by a reduction in the membrane expression of the channel. Collectively, our observations support the idea that functional changes observed in the I890T currents are likely due to altered intrinsic properties of the channel. The advent of the crystal structure of NavAb [11] has provided a structural framework to model mutations in sodium channels. The homologous residue to I890Nav1.5 is T169NavAb (Fig. 6A, upper panel). This Thr is conserved in the NaChBac bacterial sodium ML-281 channel (T187NaChBac) and in the sodium channel of alphaproteobacteria HIMB114 (T172NavRh), the structure of which has recently been solved [36]. In the available structure of NavAb channel, T169NavAb is buried within the intramembrane region of the pore. The polar group of T169NavAb is stabilized by a hydrogen bond to R185NavAb of the adjacent subunit [11]. The NavRh channel stabilizes T172 in a similar fashion: the alcohol ?group of T172NavRh is within hydrogen bond distance (2.7?.2 A depending on the subunit) of the Eledoisin site carbonyl oxygens of R162 and I168 of the same chain. Interestingly, the mutation I890T recollects the bacterial residue at this position. However, in our I890T Nav1.5 model, no hydrogen bond acceptor candidate lies in the proximity of T890 and we cannot predict how the introduction of this polar group might be stabilized. It is interesting to note that isoleucine is the aminoacid present in position 890 in Nav1.5 as well as in the homologous position of different sodium channels in a wide variety of vertebrates (Figure S1). Thus, although the precise role of I890 in Nav1.5 is unknown, its presence at this position has been evolutionary favored. In summary, we have identified a Nav1.5 pore mutation, I890T, in a BrS 
patient. This novel mutation causes an evident reduction in INa and a depolarizing shift in current voltage-dependent activation. Both mutation-dependent effects create the conditions for the observed pathophysiological manifestations of the patient. Although the observed changes in channel function in the mutated protein are mild, we cannot exclude that the effects of the mutation I890T 1326631 in native myocytes could be different from those observed in our HEK cell experimental model, due to different regulatory factors. In addition, our functional study is analyzed in the context of the recently published crystal structure of the bacterial NavAb and NavRh channels. The evident, although mild, functional effect of the mutation correlates well with the lack of major structural changes found in the in silico analysis. In this sense, we believe that this type of studies hold the promise that correlations among channel structure, functional effects of mutations and clinical studies can lead to better understanding of SCD-related channelopathies.?resolution 2.7 and 3.05 A, respectively) to model the pore domain of DII in Nav1.5. Amino acid sequences were only moderately conserved in S5 6 of DII in Nav1.5 compared to NavRh (17 identity and 25 homology) and NavAb (22 identity and 46 homology). Consequently, at the resolution of the model, i.S. It has been previously shown that mutations in the pore region may affect voltage activation of Nav1.5 and other Nav channels [32?5]. The hypothesis that mutations in the pore region of Nav channels can lead to structural perturbation compromising activation has been put forward [32]. In this context, our immunodetection experiments indicated that the decrease in INa observed in I890T cells could not be explained by a reduction in the membrane expression of the channel. Collectively, our observations support the idea that functional changes observed in the I890T currents are likely due to altered intrinsic properties of the channel. The advent of the crystal structure of NavAb [11] has provided a structural framework to model mutations in sodium channels. The homologous residue to I890Nav1.5 is T169NavAb (Fig. 6A, upper panel). This Thr is conserved in the NaChBac bacterial sodium channel (T187NaChBac) and in the sodium channel of alphaproteobacteria HIMB114 (T172NavRh), the structure of which has recently been solved [36]. In the available structure of NavAb channel, T169NavAb is buried within the intramembrane region of the pore. The polar group of T169NavAb is stabilized by a hydrogen bond to R185NavAb of the adjacent subunit [11]. The NavRh channel stabilizes T172 in a similar fashion: the alcohol ?group of T172NavRh is within hydrogen bond distance (2.7?.2 A depending on the subunit) of the carbonyl oxygens of R162 and I168 of the same chain. Interestingly, the mutation I890T recollects the bacterial residue at this position. However, in our I890T Nav1.5 model, no hydrogen bond acceptor candidate lies in the proximity of T890 and we cannot predict how the introduction of this polar group might be stabilized. It is interesting to note that isoleucine is the aminoacid present in position 890 in Nav1.5 as well as in the homologous position of different sodium channels in a wide variety of vertebrates (Figure S1). Thus, although the precise role of I890 in Nav1.5 is unknown, its presence at this position has been evolutionary favored. In summary, we have identified a Nav1.5 pore mutation, 
I890T, in a BrS patient. This novel mutation causes an evident reduction in INa and a depolarizing shift in current voltage-dependent activation. Both mutation-dependent effects create the conditions for the observed pathophysiological manifestations of the patient. Although the observed changes in channel function in the mutated protein are mild, we cannot exclude that the effects of the mutation I890T 1326631 in native myocytes could be different from those observed in our HEK cell experimental model, due to different regulatory factors. In addition, our functional study is analyzed in the context of the recently published crystal structure of the bacterial NavAb and NavRh channels. The evident, although mild, functional effect of the mutation correlates well with the lack of major structural changes found in the in silico analysis. In this sense, we believe that this type of studies hold the promise that correlations among channel structure, functional effects of mutations and clinical studies can lead to better understanding of SCD-related channelopathies.?resolution 2.7 and 3.05 A, respectively) to model the pore domain of DII in Nav1.5. Amino acid sequences were only moderately conserved in S5 6 of DII in Nav1.5 compared to NavRh (17 identity and 25 homology) and NavAb (22 identity and 46 homology). Consequently, at the resolution of the model, i.
