Mechanism of Loss of Kv11.1 K (cid:1) Current in Mutant T421M-Kv11.1–Expressing Rat Ventricular Myocytes Interaction of Trafficking and Gating

Background —Type 2 long QT syndrome involves mutations in the human ether a-go-go–related gene ( hERG or KCNH2 ). T421M, an S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking, and pharmacological mechanisms in adult rat ventricular myocytes. Methods and Results —Isolated adult rat ventricular myocytes were infected with wild-type (WT)–Kv11.1– and T421M-Kv11.1–expressing adenovirus and analyzed with the use of patch clamp, Western blot, and confocal imaging techniques. Expression of WT-Kv11.1 or T421M-Kv11.1 produced peak tail current ( I Kv11.1 ) of 8.78 (cid:1) 1.18 and 1.91 (cid:1) 0.22 pA/pF, respectively. Loss of mutant I Kv11.1 resulted from (1) a partially trafficking-deficient channel protein with reduced cell surface expression and (2) altered channel gating with a positive shift in the voltage dependence of activation and altered kinetics of activation and deactivation. Coexpression of WT (cid:2) T421M-Kv11.1 resulted in heterotetrameric channels that remained partially trafficking deficient with only a minimal increase in peak I Kv11.1 density, whereas the voltage dependence of channel gating became WT-like. In the adult rat ventricular myocyte model, both WT-Kv11.1 and T421M-Kv11.1 channels responded to (cid:1) -adrenergic stimulation by increasing I Kv11.1 . Conclusions —The T421M-Kv11.1 mutation caused a loss of I Kv11.1 through interactions of abnormal protein trafficking and channel gating. Furthermore, for coexpressed WT (cid:2) T421M-Kv11.1 channels, different dominant-negative interactions govern protein trafficking and ion channel gating, and these are likely to be reflected in the clinical phenotype. Our results also show that WT and mutant Kv11.1 channels responded to (cid:1) -adrenergic stimulation. ( Circulation . 2012;126:2809-2818.) S1 domain mutation should alter both protein trafficking and gating properties of Kv11.1 channels. We overexpressed channels in a native adult rat ventricular myocyte (ARVM) model, and we studied WT-Kv11.1 or T421M-Kv11.1 homotetrameric channels as well as coexpressing genes for WT (cid:2) T421M-Kv11.1 subunits to create heterotetrameric channels that more closely simulate human heart. Because the patient experienced cardiac arrest after an acute auditory trigger, we also studied whether there was a link between (cid:1) -adrenergic stimulation and Kv11.1 channel activity. These studies demonstrate the feasibility of using ARVMs as a native myocyte model for Kv11.1 expression and regulation studies, and the complex mechanisms we found are likely to contribute to determin-Received or T421M Kv11.1 protein (AdT421M-Kv11.1) or green fluorescent protein (AdGFP) and, for coexpression studies, 0.5 (cid:4) 10 6 of AdWT-Kv11.1 and 0.5 (cid:4) 10 6 of AdT421M-Kv11.1 viral particles on the day of isolation for 24 hours, and experiments were conducted after 4 days of infection once the expression of GFP was confirmed. (cid:1) -adrenergic stimulation of wild-type–Kv11.1 and channels in adult ventricular myocytes. Wild-type–Kv11.1 and T421M-Kv11.1 channels responded to by increasing could a direct substrate for (cid:1) -blocker therapy. Our findings provide new into