Gui J , Wang T , Jones RP , Trump D , Zimmer T , Lei M .

081283 Wellcome Trust , 081809 Wellcome Trust , pG/08/006/24399 British Heart Foundation , Wellcome Trust

	Figure 1. Representative HEK293 whole-cell current recordings for hNav1.5 and SSS-associated mutants.(A) Group 1 mutants produced peak current densities similar to wild-type hNav1.5. (B) Group 2 mutants produced reduced currents. (C) Group 3 mutants produced no current. Table 1 shows average peak current densities and statistics thereof.
	Figure 2. Electrophysiological properties of four group 1 mutant channels expressed by HEK293 cells.Individual parameters are summarized in Table 1. (A) Peak current-to-voltage (I–V) relationships. Currents were elicited from the holding potential of −120 mV to the test potentials indicated. (B) Steady-state activation as a function of voltage. (C) Steady-state inactivation as a function of voltage. (D) Recovery from inactivation. Normalized data were fitted to double exponentials yielding the fast and slow time constants listed in Table 1. (E, F) Time constant of inactivation (τh) as a function of voltage. Parameters were obtained from monoexponential fits. Error bars represent the mean ± SEM.
	Figure 3. Electrophysiological properties of three group 2 mutant channels expressed by HEK293 cells.Individual parameters are summarized in Table 1. (A) Peak current-to-voltage (I–V) relationships. Currents were elicited from the holding potential of −120 mV to the indicated test potentials. (B) Steady-state activation as a function of voltage. (C) Steady-state inactivation as a function of voltage. (D) Recovery from inactivation. (E) Time constant of inactivation (τh) as function of voltage. Error bars represent the mean ± SEM.
	Figure 4. A comparison of normalized peak current amplitudes for wild-type and mutant hNav1.5 channels expressed by two different expression systems.Whole-cell currents were recorded by the patch clamp technique (HEK293 cells) and by the two-microelectrode voltage clamp technique (oocytes) as described in “Methods”. L212P and T220I exhibited similar relative expression levels in both systems. With E161K, D1275N, and P1298L, whole-cell currents, normalized to hNav1.5 values, were significantly smaller for the oocyte system (* indicates p<0.01). Channels classified as group 3 mutants upon expression by HEK293 cells (T187I, R878C, G1408R, W1421X) did not produce detectable Na+ inward currents in the oocyte system, even upon injection of undiluted cRNA (data not shown).
	Figure 5. Representative Western blots showing Nav1.5 polypeptides purified from the surface of HEK293 cells by the biotinylation/precipitation procedure (cell surface) and the Nav1.5 polypeptides in HEK293 cell lysates (total).Calnexin was used as a control to demonstrate the absence of endoplasmic reticulum membranes in the isolated plasma membrane fraction.
	Figure 6. Correlation between cell surface expression and peak current densities for the thirteen SSS-associated mutant channels.Normalized cell surface signals were obtained by dividing the “cell surface” signal by the corresponding “total” signal; both signals being obtained from the same Western blot (see Fig. 5). A ratio obtained for a mutant was further normalized by dividing with the ratio obtained for wild-type hNav1.5 again from the same blot. The mean double-normalized cell surface signals shown were calculated from at least three independent biotinylation experiments. * indicates p<0.05, ** indicates p<0.01.
	Figure 7. Summary of the molecular mechanisms underlying SCN5A-related familial SSS.(A) Proposed membrane topology of hNav1.5 showing the locations of the thirteen SSS-associated mutations investigated here. Compared to wild-type hNav1.5, the mutant channels produced either similar (group 1, white), reduced (group 2, light grey) or no (group 3, dark grey) current. (B) Schematic representation of a sinus node cell and the cellular mechanisms involved in SCN5A-related familial SSS. Early onset of familial SSS occurred (a) when two alleles were affected (compound mutations P1298L/G1408R, DelF1617/R1632H, and T220I/R1623X) [16], or (b) when other risk factors were present (for example, a Cx40 polymorphism combined with L212P; Table 3) [34]. Notably, when wild-type hNav1.5 channels are expressed from the normal allele, most of the mutants also caused other cardiac disorders like BrS, AV block, CCD or DCM, as observed for carriers of T187I, W1421X, K1578fs/52, and E161K (see Discussion and Table 3). In most if not all of these cases, SSS onset relatively late.

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