Interaction of the S6 Proline Hinge with N-Type and C-Type Inactivation in Kv1.4 Channels

AbstractSeveral voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials

Investigation of the Effects of the Short QT Syndrome D172N Kir2.1 Mutation on Ventricular Action Potential Profile Using Dynamic Clamp

The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K(+) current, I(K1). The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of I(K1) using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic I(K1) was inhibited with a low concentration (50 µM) of Ba(2+) ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic I(K1) through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) I(K1) with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD(90)) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD(90) and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant I(K1) to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations

General Principles for the Validation of Proarrhythmia Risk Prediction Models: An Extension of the CiPA In Silico Strategy

This white paper presents principles for validating proarrhythmia risk prediction models for regulatory use as discussed at the In Silico Breakout Session of a Cardiac Safety Research Consortium/Health and Environmental Sciences Institute/US Food and Drug Administration–sponsored Think Tank Meeting on May 22, 2018. The meeting was convened to evaluate the progress in the development of a new cardiac safety paradigm, the Comprehensive in Vitro Proarrhythmia Assay (CiPA). The opinions regarding these principles reflect the collective views of those who participated in the discussion of this topic both at and after the breakout session. Although primarily discussed in the context of in silico models, these principles describe the interface between experimental input and model‐based interpretation and are intended to be general enough to be applied to other types of nonclinical models for proarrhythmia assessment. This document was developed with the intention of providing a foundation for more consistency and harmonization in developing and validating different models for proarrhythmia risk prediction using the example of the CiPA paradigm

Markov models of use-dependence and reverse use-dependence during the mouse cardiac action potential.

The fast component of the cardiac transient outward current, I(Ktof), is blocked by a number of drugs. The major molecular bases of I(Ktof) are Kv4.2/Kv4.3 voltage-gated potassium channels. Drugs with similar potencies but different blocking mechanisms have differing effects on action potential duration (APD). We used in silico analysis to determine the effect of I(Ktof)-blocking drugs with different blocking mechanisms on mouse ventricular myocytes. We used our existing mouse model of the action potential, and developed 4 new Markov formulations for I(Ktof), I(Ktos), I(Kur), I(Ks). We compared effects of theoretical I(Ktof)-specific channel blockers: (1) a closed state, and (2) an open channel blocker. At concentrations lower or close to IC(50), the drug which bound to the open state always had a much greater effect on APD than the drug which bound to the closed state. At concentrations much higher than IC(50), both mechanisms had similar effects at very low pacing rates. However, an open state binding drug had a greater effect on APD at faster pacing rates, particularly around 10 Hz. In summary, our data indicate that drug effects on APD are strongly dependent not only on IC(50), but also on the drug binding state