The human etherà‐go‐go‐related gene (HERG) encodes a K+o channel that is believed to be the basis of the delayed rectified current, IKr, in cardiac muscle. We studied HERG expressed in Xenopus oocytes using a two‐electrode and cut‐open oocyte clamp technique with [K+]o of 2 and 98 mm. The time course of activation of the channel was measured using an envelope of tails protocol and demonstrated that activation of the heterologously expressed HERG current (IHERG) was sigmoidal in onset. At least three closed states were required to reproduce the sigmoid time course. The voltage dependence of the activation process and its saturation at positive voltages suggested the existence of at least one relatively voltage‐insensitive step. A three closed state activation model with a single voltage‐insensitive intermediate closed state was able to reproduce the time and voltage dependence of activation, deactivation and steady‐state activation. Activation was insensitive to changes in [K+]o. Both inactivation and recovery time constants increased with a change of [K+]o from 2 to 98 mm. Steady‐state inactivation shifted by ∼30 mV in the depolarized direction with a change from 2 to 98 mm K*o Simulations showed that modulation of inactivation is a minimal component of the increase of this current by [K+]o, and that a large increase in total conductance must also occur.

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Background Human induced pluripotent stem cell (h-iPSC)-derived cardiac myocytes are a unique model in which to study human myocyte function and dysfunction, especially from patients with genetic disorders. They are also considered a major advance for drug safety testing. However, these cells have considerable unexplored potential limitations when applied to quantitative action potential (AP) analysis. One major factor is spontaneous activity, and resulting variability and potentially anomalous behavior in AP parameters. Objective To demonstrate the effect of using an in silico interface to electronically express IK1, a major component lacking in h-iPSC-derived cardiac myocytes. Methods An in silico interface was developed to express synthetic IK1 in cells under whole cell voltage clamp. Results Electronic IK1 expression established a physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed after depolarizations, and decreased AP variability. Initiated APs had the classic rapid upstroke and spike and dome morphology consistent with data from freshly isolated human myocytes, and the readily recognizable repolarization attributes of ventricular and atrial cells. Application of 1 μM BayK-8644 resulted in anomalous AP shortening in h-iPSC-derived cardiac myocytes. When IK1 was electronically expressed, BayK-8644 lengthened the AP, consistent with existing results on native cardiac myocytes. Conclusions Electronic expression of IK1 is a simple and robust method to significantly improve the physiological behavior of the AP and electrical profile of h-iPSC-derived cardiac myocytes. Increased stability enables this preparation to be used for controlled quantitative analysis of AP parameters e.g., drug responsiveness, genetic disorders, and dynamic behavior restitution profiles.

1. A fast inactivating transient K+ current (FK1) cloned from ferret ventricle and expressed in Xenopus oocytes was studied using the two-electrode voltage clamp technique. Removal of the NH2-terminal domain of FK1 (FK1A2-146) removed fast inactivation consistent with previous findings in Kv1.4 channels. The NH2-terminal deletion mutation revealed a slow inactivation process, which matches the criteria for C-type inactivation described for Shaker B channels. 2. Inactivation of FK1A2-146 at depolarized potentials was well described by a single exponential process with a voltage-insensitive time constant. In the range -90 to +20 mV, steady-state C-type inactivation was well described by a Boltzmann relationship that compares closely with inactivation measured in the presence of the NH2-terminus. These results suggest that C-type inactivation is coupled to activation. 3. The coupling of C-type inactivation to activation was assessed by mutation of the fourth positively charged residue (arginine 454) in the S4 voltage sensor to glutamine (R454Q). This mutation produced a hyperpolarizing shift in the inactivation relationship of both FK1 and FK1A2-146 without altering the rate of inactivation of either clone. 4. The rates of recovery from inactivation are nearly identical in FK1 and FK1A2-146. 5. To assess the mechanisms underlying recovery from inactivation the effects of elevated [K+]. and selective mutations in the extracellular pore and the S4 voltage sensor were compared in FK1 and FK1A2-146. The similarity in recovery rates in response to these perturbations suggests that recovery from C-type inactivation governs the overall rate of recovery of inactivated channels for both . Analysis of the rate of recovery of FK1 channels for inactivating pulses of different durations (70-2000 ms) indicates that recovery rate is insensitive to the duration of the inactivating pulse.

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