Non-technical summary Diseases, genetic defects, or ionic imbalances can alter the normal electrical activity of cardiac myocytes causing an anomalous heart rhythm, which can degenerate to ventricular fibrillation (VF) and sudden cardiac death. Well-recognized triggers for VF are aberrations of the cardiac action potential, known as early afterdepolarizations (EADs). In this study, combining mathematical modelling and experimental electrophysiology in real-time (dynamic clamp), we investigated the dependence of EADs on the biophysical properties of the L-type Ca 2+ current (I Ca,L ) and identified modifications of I Ca,L properties which effectively suppress EAD. We found that minimal changes in the voltage dependence of activation or inactivation of I Ca,L can dramatically reduce the occurrence of EADs in cardiac myocytes exposed to different EAD-inducing conditions. This work assigns a critical role to the L-type Ca 2+ channel biophysical properties for EADs formation and identifies the L-type Ca 2+ channel as a promising therapeutic target to suppress EADs and their arrhythmogenic effects.Abstract Sudden cardiac death (SCD) due to ventricular fibrillation (VF) is a major world-wide health problem. A common trigger of VF involves abnormal repolarization of the cardiac action potential causing early afterdepolarizations (EADs). Here we used a hybrid biological-computational approach to investigate the dependence of EADs on the biophysical properties of the L-type Ca 2+ current (I Ca,L ) and to explore how modifications of these properties could be designed to suppress EADs. EADs were induced in isolated rabbit ventricular myocytes by exposure to 600 μmol l −1 H 2 O 2 (oxidative stress) or lowering the external [K + ] from 5.4 to 2.0-2.7 mmol l −1 (hypokalaemia). The role of I Ca,L in EAD formation was directly assessed using the dynamic clamp technique: the paced myocyte's V m was input to a myocyte model with tunable biophysical parameters, which computed a virtual I Ca,L , which was injected into the myocyte in real time. This virtual current replaced the endogenous I Ca,L , which was suppressed with nifedipine. Injecting a current with the biophysical properties of the native I Ca,L restored EAD occurrence in myocytes challenged by H 2 O 2 or hypokalaemia. A mere 5 mV depolarizing shift in the voltage dependence of activation or a hyperpolarizing shift in the steady-state inactivation curve completely abolished EADs in myocytes while maintaining a normal Ca i transient. We propose that modifying the biophysical properties of I Ca,L has potential as a powerful therapeutic strategy for suppressing EADs and EAD-mediated arrhythmias.

Decreasing the amplitude of the late component of the cardiac L-type Ca2+ channel current suppresses early afterdepolarizations, thereby decreasing the risk of arrhythmias.

To complete a study on site-directed alkylation of Cys replacements in the lactose permease of Escherichia coli (LacY), the reactivity of single-Cys mutants in helices I, III, VI, and XI, as well as some of the adjoining loops, with N-[14C]ethylmaleimide (NEM) or methanethiosulfonate ethylsulfonate (MTSES) was studied in right-side-out membrane vesicles. With the exception of several positions in the middle of helix I, which either face the bilayer or are in close proximity to other helices, the remaining Cys replacements react with the membrane-permeant alkylating agent NEM. In helices III and XI, most Cys replacements are also alkylated by NEM except for positions that face the bilayer. The reactivity of Cys replacements in helix VI is noticeably lower and only 45% of the replacements label. Binding of sugar leads to significant increases in the reactivity of Cys residues that are located primarily at the same level as the sugar-binding site or in the periplasmic half of each helix. Remarkably, studies with small, impermeant MTSES show that single-Cys replacements in the cytoplasmic portions of helices I and XI, which line the inward-facing cavity, are accessible to solvent from the periplasmic surface of the membrane. Moreover, addition of ligand results in increased accessibility of Cys residues to the aqueous milieu in the periplasmic region of the helices, which may reflect structural rearrangements leading to opening of an outward-facing cavity. The findings are consistent with the X-ray structure of LacY and with the alternating access model [Abramson, J., Smirnova, I., et al. (2003) Science 301, 610-615].