Automated Patch ClampingCells were patch clamped with the SyncroPatch 384PE automated patch clamping device (Nanion). To prepare cells for patch clamping, cells were washed in PBS, treated with Accutase (Millipore-Sigma) for 3 min at 37°C, then recovered in CHO-S-serum free media (GIBCO). Cells were pelleted and resuspended in divalent-free reference solution (DVF) at ∼200,000–400,000 cells/mL. DVF contained (mM) NaCl 140, KCl 4, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with NaOH. Cells were then added to a medium resistance (4–6 MΩ) 384-well recording chamber with 1 patch aperture per well (NPC-384, Nanion), which contained DVF and internal solution: CsCl 10, NaCl 10, CsF 110, EGTA 10, HEPES 10 (pH 7.2) adjusted with CsOH. Next, to enhance seal resistance, 50% of the DVF was exchanged with a calcium-containing seal enhancing solution: NaCl 80, NMDG 60, KCl 4, MgCl2 1, CaCl2 10, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with HCl. The cells were washed three times in external recording solution: NaCl 80, NMDG 60, KCl 4, MgCl2 1, CaCl2 2, alpha-D(+)-glucose 5, HEPES 10 (pH 7.4) adjusted with HCl. Currents elicited in response to activation, inactivation, and recovery from inactivation protocols were then recorded (Figure S2). A late current measurement was captured every 5 s. After 1 min, 50% of the external solution was exchanged with external solution containing 200 μM tetracaine hydrochloride (Sigma; effective concentration 100 μM tetracaine). After tetracaine addition, late current measurements were obtained every 5 s for 1 additional minute. At least 10 cells expressing wild-type SCN5A were included for comparison in each SyncroPatch experiment (Figure 1), and at least 2 independent transfections and at least 10 replicate cells were studied per mutant. Recordings were performed at room temperature.We also conducted experiments to assess the effects of incubation at low temperature or mexiletine (a sodium channel blocker), interventions reported to increase cell surface expression of mistrafficked channels.27Clatot J. Ziyadeh-Isleem A. Maugenre S. Denjoy I. Liu H. Dilanian G. Hatem S.N. Deschênes I. Coulombe A. Guicheney P. Neyroud N. Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Na(v)1.5 α-subunits.Cardiovasc. Res. 2012; 96: 53-63Crossref PubMed Scopus (62) Google Scholar,  28Makiyama T. Akao M. Tsuji K. Doi T. Ohno S. Takenaka K. Kobori A. Ninomiya T. Yoshida H. Takano M. et al.High risk for bradyarrhythmic complications in patients with Brugada syndrome caused by SCN5A gene mutations.J. Am. Coll. Cardiol. 2005; 46: 2100-2106Crossref PubMed Scopus (99) Google Scholar,  29Pfahnl A.E. Viswanathan P.C. Weiss R. Shang L.L. Sanyal S. Shusterman V. Kornblit C. London B. Dudley Jr., S.C. A sodium channel pore mutation causing Brugada syndrome.Heart Rhythm. 2007; 4: 46-53Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,  30Valdivia C.R. Ackerman M.J. Tester D.J. Wada T. McCormack J. Ye B. Makielski J.C. A novel SCN5A arrhythmia mutation, M1766L, with expression defect rescued by mexiletine.Cardiovasc. Res. 2002; 55: 279-289Crossref PubMed Scopus (77) Google Scholar,  31Valdivia C.R. Tester D.J. Rok B.A. Porter C.B. Munger T.M. Jahangir A. Makielski J.C. Ackerman M.J. A trafficking defective, Brugada syndrome-causing SCN5A mutation rescued by drugs.Cardiovasc. Res. 2004; 62: 53-62Crossref PubMed Scopus (106) Google Scholar For these experiments, cells stably expressing loss-of-function variants were generated as described above. The cells were incubated for 24 h at 30°C, or at 37°C with or without 500 μM mexiletine hydrochloride (Sigma), washed with HEK media, and were patch clamped as described above.