Assessment of proarrhythmogenic risk for chloroquine and hydroxychloroquine using the CiPA concept.

Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of µM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the ß-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.

Identification of structures for ion channel kinetic models.

Markov models of ion channel dynamics have evolved as experimental advances have improved our understanding of channel function. Past studies have examined limited sets of various topologies for Markov models of channel dynamics. We present a systematic method for identification of all possible Markov model topologies using experimental data for two types of native voltage-gated ion channel currents: mouse atrial sodium currents and human left ventricular fast transient outward potassium currents. Successful models identified with this approach have certain characteristics in common, suggesting that aspects of the model topology are determined by the experimental data. Incorporating these channel models into cell and tissue simulations to assess model performance within protocols that were not used for training provided validation and further narrowing of the number of acceptable models. The success of this approach suggests a channel model creation pipeline may be feasible where the structure of the model is not specified a priori.

Regulation of membrane KCNQ1/KCNE1 channel density by sphingomyelin synthase 1.

Sphingomyelin synthase (SMS) catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol. We previously showed that SMS1 deficiency leads to a reduction in expression of the K(+) channel KCNQ1 in the inner ear (Lu MH, Takemoto M, Watanabe K, Luo H, Nishimura M, Yano M, Tomimoto H, Okazaki T, Oike Y, and Song WJ. J Physiol 590: 4029-4044, 2012), causing hearing loss. However, it remains unknown whether this change in expression is attributable to a cellular process or a systemic effect in the knockout animal. Here, we examined whether manipulation of SMS1 activity affects KCNQ1/KCNE1 currents in individual cells. To this end, we expressed the KCNQ1/KCNE1 channel in human embryonic kidney 293T cells and evaluated the effect of SMS1 manipulations on the channel using whole cell recording. Application of tricyclodecan-9-yl-xanthogenate, a nonspecific inhibitor of SMSs, significantly reduced current density and altered channel voltage dependence. Knockdown of SMS1 by a short hairpin RNA, however, reduced current density alone. Consistent with this, overexpression of SMS1 increased the current density without changing channel properties. Furthermore, application of protein kinase D inhibitors also suppressed current density without changing channel properties; this effect was nonadditive with that of SMS1 short hairpin RNA. These results suggest that SMS1 positively regulates KCNQ1/KCNE1 channel density in a protein kinase D-dependent manner.

Comparison of Langevin and Markov channel noise models for neuronal signal generation.

The stochastic opening and closing of voltage-gated ion channels produce noise in neurons. The effect of this noise on the neuronal performance has been modeled using either an approximate or Langevin model based on stochastic differential equations or an exact model based on a Markov process model of channel gating. Yet whether the Langevin model accurately reproduces the channel noise produced by the Markov model remains unclear. Here we present a comparison between Langevin and Markov models of channel noise in neurons using single compartment Hodgkin-Huxley models containing either Na+ and K+, or only K+ voltage-gated ion channels. The performance of the Langevin and Markov models was quantified over a range of stimulus statistics, membrane areas, and channel numbers. We find that in comparison to the Markov model, the Langevin model underestimates the noise contributed by voltage-gated ion channels, overestimating information rates for both spiking and nonspiking membranes. Even with increasing numbers of channels, the difference between the two models persists. This suggests that the Langevin model may not be suitable for accurately simulating channel noise in neurons, even in simulations with large numbers of ion channels.

In vitro assessment of the neurotoxic and neuroprotective effects of N-acetyl-L-cysteine (NAC) on the rat sciatic nerve fibers.

N-acetyl-L-cysteine (NAC), at a concentration of 1-60mM, has been previously used extensively for protection in a variety of cell cultures against the deleterious effects of various compounds. The results of this in vitro study show that NAC has certain unusual effects on the evoked compound action potential (CAP) of the rat sciatic nerve fibers. Firstly, at concentrations of 5.0, 3.5 and 2.5mM, concentrations used by others as a protectant for cell cultures, NAC inhibits the action potentials of the sciatic nerve fibers completely in a concentration-dependent manner within a few minutes or hours (2.5mM). Secondly, the acute inhibitory action of NAC on the CAP of the nerve fibers was not spontaneously reversible, but as soon as NAC was replaced with saline there was a partial (approximately 75%) recovery in the function of the nerve fibers. Thirdly, the no observed effect concentration for NAC was estimated to be 1mM. The paradox is that NAC at 1 mM not only had no effect on the nerve fibers, but it became an excellent neuroprotective compound, giving almost 100% neuroprotection against cadmium-induced neurotoxicity. The results show a possible effect of NAC on voltage-gated sodium and potassium channels. The observed neuroprotective-neurotoxic properties of NAC require careful reconsideration of its use in either in vitro studies or in vivo pharmaceutical applications.

Activation properties of Kv4.3 channels: time, voltage and [K+]o dependence.

Rapidly inactivating, voltage-dependent K(+) currents play important roles in both neurones and cardiac myocytes. Kv4 channels form the basis of these currents in many neurones and cardiac myocytes and their mechanism of inactivation appears to differ significantly from that reported for Shaker and Kv1.4 channels. In most channel gating models, inactivation is coupled to the kinetics of activation. Hence, there is a need for a rigorous model based on comprehensive experimental data on Kv4.3 channel activation. To develop a gating model of Kv4.3 channel activation, we studied the properties of Kv4.3 channels in Xenopus oocytes, without endogenous KChIP2 ancillary subunits, using the perforated cut-open oocyte voltage clamp and two-electrode voltage clamp techniques. We obtained high-frequency resolution measurements of the activation and deactivation properties of Kv4.3 channels. Activation was sigmoid and well described by a fourth power exponential function. The voltage dependence of the activation time constants was best described by a biexponential function corresponding to at least two different equivalent charges for activation. Deactivation kinetics are voltage dependent and monoexponential. In contrast to other voltage-sensitive K(+) channels such as HERG and Shaker, we found that elevated extracellular [K(+)] modulated the activation process by slowing deactivation and stabilizing the open state. Using these data we developed a model with five closed states and voltage-dependent transitions between the first four closed states coupled to a voltage-insensitive transition between the final closed (partially activated) state and the open state. Our model closely simulates steady-state and kinetic activation and deactivation data.