Michael J. Morales

A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes.

1 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. 2 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. 3 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. 4 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 5 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.

In Situ Hybridization Reveals Extensive Diversity of K+ Channel mRNA in Isolated Ferret Cardiac Myocytes

The molecular basis of K+ currents that generate repolarization in the heart is uncertain. In part, this reflects the similar functional properties different K+ channel clones display when heterologously expressed, in addition to the molecular diversity of the voltage-gated K+ channel family. To determine the identity, regional distribution, and cellular distribution of voltage-sensitive K+ channel mRNA subunits expressed in ferret heart, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes from the sinoatrial (SA) node, right and left atria, right and left ventricles, and interatrial and interventricular septa. The most widely distributed K+ channel transcripts in the ferret heart were Kv1.5 (present in 69.3% to 85.6% of myocytes tested, depending on the anatomic region from which myocytes were isolated) and Kv1.4 (46.1% to 93.7%), followed by kv1.2, Kv2.1, and Kv4.2. Surprisingly, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1, which were rarely expressed in working myocytes, were more commonly expressed in SA nodal cells. IRK was expressed in ventricular (84.3% to 92.8%) and atrial (52.4% to 64.0%) cells but was nearly absent (6.6%) in SA nodal cells; minK was most frequently expressed in SA nodal cells (33.7%) as opposed to working myocytes (10.3% to 29.3%). Two gene products implicated in long-QT syndrome, ERG and KvLQT1, were common in all anatomic regions (41.1% to 58.2% and 52.1% to 71.8%, respectively). These results show that the diversity of K+ channel mRNA in heart is greater than previously suspected and that the molecular basis of K+ channels may vary from cell to cell within distinct regions of the heart and also between major anatomic regions.

Regional Localization of ERG, the Channel Protein Responsible for the Rapid Component of the Delayed Rectifier, K+ Current in the Ferret Heart

Repolarization of the cardiac action potential varies widely throughout the heart. This could be due to the differential distribution of ion channels responsible for repolarization, especially the K+ channels. We have therefore studied the cardiac localization of ERG, a channel protein known to play an important role in generation of the rapid component of the delayed rectifier K+ current (IKr), an important determinant of the repolarization waveform, Cryosections of the ferret atrium and ventricle were prepared to determine the localization of ERG by fluorescence in situ hybridization (FISH) and immunofluorescence. We found that in the ferret, ERG transcript and protein expression was most abundant in the epicardial cell layers throughout most of the ventricle, except at the base. In the atrium, we found that ERG is most abundant in the medial right atrium, especially in the trabeculae and the crista terminalis of the right atrial appendage. It also is present in areas within the sinoatrial node. In all regions studied, FISH and immunofluorescence showed concordant localization patterns. These data suggest that repolarization mediated by IKr is not uniform throughout the ferret heart and provide a molecular explanation for heterogeneity in action potential repolarization throughout the mammalian heart.

C-type inactivation controls recovery in a fast inactivating cardiac K+ channel (Kv1.4) expressed in Xenopus oocytes.

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 (FK1 delta 2‐146) 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 FK1 delta 2‐146 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 FK1 delta 2‐146 without altering the rate of inactivation of either clone. 4. The rates of recovery from inactivation are nearly identical in FK1 and FK1 delta 2‐146. 5. To assess the mechanisms underlying recovery from inactivation the effects of elevated [K+]o and selective mutations in the extracellular pore and the S4 voltage sensor were compared in FK1 and FK1 delta 2‐146. 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 FK1 and FK1 delta 2‐146. 6. 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.

Time, voltage and ionic concentration dependence of rectification of h-erg expressed in Xenopus oocytes

The rapid delayed rectifier, IKr is believed to have h‐erg ( uman ther‐à‐go‐go elated ene) as its molecular basis. A recent study has shown that rectification of h‐erg involves a rapid inactivation process that involves rapid closure of the external mouth of the pore or C‐type inactivation. We measured the instantaneous current to voltage relationship for h‐erg channels using the saponin permeabilized variation of the cut‐open oocyte clamp technique. In contrast to C‐type inactivation in other voltage‐gated K+ channels, the rate of inactivation was strongly voltage dependent at depolarized potentials. This voltage dependence could be modulated independently of activation by increasing [K+]o from 2 to 98 mM. These results suggest that inactivation of h‐erg has its own intrinsic voltage sensor.

Heterogeneous expression of KChIP2 isoforms in the ferret heart

Kv4 channels are believed to underlie the rapidly recovering cardiac transient outward current (Ito) phenotype. However, heterologously expressed Kv4 channels fail to fully reconstitute the native current. Kv channel interacting proteins (KChIPs) have been shown to modulate Kv4 channel function. To determine the potential involvement of KChIPs in the rapidly recovering Ito, we cloned three KChIP2 isoforms (designated fKChIP2, 2a and 2b) from the ferret heart. Based upon immunoblot data suggesting the presence of a potential endogenous KChIP‐like protein in HEK 293, CHO and COS cells but absence in Xenopus oocytes, we coexpressed Kv4.3 and the fKChIP2 isoforms in Xenopus oocytes. Functional analysis showed that while all fKChIP2 isoforms produced a fourfold acceleration of recovery kinetics compared to Kv4.3 expressed alone, only fKChIP2a produced large depolarizing shifts in the V1/2 of steady‐state activation and inactivation as seen for the native rapidly recovering Ito. Analysis of RNA and protein expression of the three fKChIP2 isoforms in ferret ventricles showed that fKChIP2b was most abundant and was expressed in a gradient paralleling the rapidly recovering Ito distribution. Ferret KChIP2 and 2a were expressed at very low levels. The ventricular expression distribution suggests that fKChIP2 isoforms are involved in modulation of the rapidly recovering Ito; however, additional regulatory factors are also likely to be involved in generating the native current.

Electronic “expression” of the inward rectifier in cardiocytes derived from human-induced pluripotent stem cells

BACKGROUND Human-induced pluripotent stem cell (h-iPSC)-derived cardiac myocytes are a unique model in which human myocyte function and dysfunction are studied, especially those 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 of AP parameters. OBJECTIVE To demonstrate the effect of using an in silico interface on electronically expressed I(K1), a major component lacking in h-iPSC-derived cardiac myocytes. METHODS An in silico interface was developed to express synthetic I(K1) in cells under whole-cell voltage clamp. RESULTS Electronic I(K1) expression established a physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed afterdepolarizations, and decreased AP variability. The initiated APs had the classic rapid upstroke and spike and dome morphology consistent with data obtained with freshly isolated human myocytes as well as the readily recognizable repolarization attributes of ventricular and atrial cells. The application of 1 µM of BayK-8644 resulted in anomalous AP shortening in h-iPSC-derived cardiac myocytes. When I(K1) was electronically expressed, BayK-8644 prolonged the AP, which is consistent with the existing results on native cardiac myocytes. CONCLUSIONS The electronic expression of I(K1) 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 the use of this preparation for a controlled quantitative analysis of AP parameters, for example, drug responsiveness, genetic disorders, and dynamic behavior restitution profiles.

Kv1.4 channel block by quinidine: evidence for a drug‐induced allosteric effect

We studied quinidine block of Kv1.4ΔN, a K+ channel lacking N‐type inactivation, expressed in Xenopus ooctyes. Initially, quinidine intracellularly blocked the open channel so rapidly it overlapped with activation. This rapid open channel block was reduced (non‐additively) by interventions that slow C‐type inactivation: [K+]o elevation and an extracellular lysine to tyrosine mutation (K532Y). These manipulations reduced the affinity of rapid open channel block ≈10‐fold, but left the effective electrical distance unchanged at ≈0.15. Following rapid open channel block, there were time‐dependent quinidine effects: the rate of inactivation during a single depolarisation was increased, and repetitive pulsing showed use dependence. The rate of recovery from the time‐dependent aspect of quinidine block was similar to recovery from normal C‐type inactivation. Manipulations that prevented the channel from entering the C‐type inactivated state (i.e. high [K+]o or the K532Y mutation) prevented the development of the time‐dependent quinidine‐induced inactivation. The concentration dependence of the rapid block and the time‐dependent quinidine‐induced inactivation were similar, but the time‐dependent component was strongly voltage sensitive, with an effective electrical distance of 2. Clearly, this cannot reflect the permeation of quinidine through the electric field, but must be the result of some other voltage‐sensitive change in the channel. We propose that quinidine promotes the entry of the channel into a C‐type inactivated state in a time‐ and voltage‐dependent manner. We developed a mathematical model based on these results to test the hypothesis that, following rapid open channel block, quinidine promotes development of the C‐type inactivated state through a voltage‐dependent conformational change.

Ancillary subunits and stimulation frequency determine the potency of chromanol 293B block of the KCNQ1 potassium channel

KCNQ1 (Kv7.1 or KvLQT1) encodes the alpha‐subunit of a voltage‐gated potassium channel found in tissues including heart, brain, epithelia and smooth muscle. Tissue‐specific characteristics of KCNQ1 current are diverse, due to modification by ancillary subunits. In heart, KCNQ1 associates with KCNE1 (MinK), producing a slowly activating voltage‐dependent channel. In epithelia, KCNQ1 co‐assembles with KCNE3 (Mirp2) producing a constitutively open channel. Chromanol 293B is a selective KCNQ1 blocker. We studied drug binding and frequency dependence of 293B on KCNQ1 and ancillary subunits expressed in Xenopus oocytes. Ancillary subunits altered 293B potency up to 100‐fold (IC50 for KCNQ1 = 65.4 ± 1.7 μm; KCNQ1/KCNE1 = 15.1 ± 3.3 μm; KCNQ1/KCNE3 = 0.54 ± 0.18 μm). Block of KCNQ1 and KCNQ1/KCNE3 was time independent, but 293B altered KCNQ1/KCNE1 activation. We therefore studied frequency‐dependent block of KCNQ1/KCNE1. Repetitive rapid stimulation increased KCNQ1/KCNE1 current biphasically, and 293B abolished the slow component. KCNQ1/KCNE3[V72T] activates slowly with a KCNQ1/KCNE1‐like phenotype, but retains the high affinity binding of KCNQ1/KCNE3, demonstrating that subunit‐mediated changes in gating can be dissociated from subunit‐mediated changes in affinity. This study demonstrates the KCNQ1 pharmacology is significantly altered by ancillary subunits. The response of KCNQ1 to specific blockers will therefore be critically dependent on the electrical stimulation pattern of the target organ. Furthermore, the dissociation between gating and overall affinity suggests that mutations in ancillary subunits can potentially strongly alter drug sensitivity without obvious functional changes in gating behaviour, giving rise to unexpected side‐effects such as a predisposition to acquired long QT syndrome.

Kinetic properties of Kv4.3 and their modulation by KChIP2b.

KChIPs are a family of Kv4 K(+) channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K(+) channels.