Loose Coupling between the Voltage Sensor and the Activation Gate in Mammalian HCN Channels Suggests a Gating Mechanism.

Voltage-gated potassium (Kv) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels share similar structures but have opposite gating polarity. Kv channels have a strong coupling (>109) between the voltage sensor (S4) and the activation gate: when S4s are activated, the gate is open to >80% but, when S4s are deactivated, the gate is open <10-9 of the time. Using noise analysis, we show that the coupling between S4 and the gate is <200 in HCN channels. In addition, using voltage clamp fluorometry, locking the gate open in a Kv channel drastically altered the energetics of S4 movement. In contrast, locking the gate open or decreasing the coupling between S4 and the gate in HCN channels had only minor effects on the energetics of S4 movement, consistent with a weak coupling between S4 and the gate. We propose that this loose coupling is a prerequisite for the reversed voltage gating in HCN channels.

Quantitative Profiling of the Effects of Vanoxerine on Human Cardiac Ion Channels and its Application to Cardiac Risk.

Vanoxerine has been in clinical trials for Parkinsonism, depression and cocaine addiction but lacked efficacy. Although a potent blocker of hERG, it produced no serious adverse events. We attributed the unexpected result to offsetting Multiple Ion Channel Effects (MICE). Vanoxerine's effects were strongly frequency-dependent and we repositioned it for treatment of atrial fibrillation and flutter. Vanoxerine terminated AF/AFL in an animal model and a dose-ranging clinical trial. Reversion to normal rhythm was associated with QT prolongation yet absent proarrhythmia markers for Torsade de Pointes (TdP). To understand the QT/TdP discordance, we used quantitative profiling and compared vanoxerine with dofetilide, a selective hERG-blocking torsadogen used for intractable AF, verapamil, a non-torsadogenic MICE comparator and bepridil, a torsadogenic MICE comparator. At clinically relevant concentrations, verapamil blocked hCav1.2 and hERG, as did vanoxerine and bepridil both of which also blocked hNav1.5. In acute experiments and simulations, dofetilide produced early after depolarizations (EADs) and arrhythmias, whereas verapamil, vanoxerine and bepridil produced no proarrhythmia markers. Of the MICE drugs only bepridil inhibited hERG trafficking following overnight exposure. The results are consistent with the emphasis on MICE of the CiPA assay. Additionally we propose that trafficking inhibition of hERG be added to CiPA.

Functional Characterization of Human Stem Cell-Derived Cardiomyocytes.

Cardiac toxicity is a leading contributor to late-stage attrition in the drug discovery process and to withdrawal of approved from the market. In vitro assays that enable earlier and more accurate testing for cardiac risk provide early stage predictive indicators that aid in mitigating risk. Human cardiomyocytes, the most relevant subjects for early stage testing, are severely limited in supply. But human stem cell-derived cardiomyocytes (SC-hCM) are readily available from commercial sources and are increasingly used in academic research, drug discovery and safety pharmacology. As a result, SC-hCM electrophysiology has become a valuable tool to assess cardiac risk associated with drugs. This unit describes techniques for recording individual currents carried by sodium, calcium and potassium ions, as well as single cell action potentials, and impedance recordings from contracting syncytia of thousands of interconnected cells.

MICE models: superior to the HERG model in predicting Torsade de Pointes.

Drug-induced block of the cardiac hERG (human Ether-à-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Nav1.5 and Cav1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Cav1.2.

Increased cardiac risk in concomitant methadone and diazepam treatment: pharmacodynamic interactions in cardiac ion channels.

Methadone, a synthetic opioid for treatment of chronic pain and withdrawal from opioid dependence, has been linked to QT prolongation, potentially fatal torsades de pointes, and sudden cardiac death. Concomitant use of diazepam or other benzodiazepines in methadone maintenance treatment can increase the risk of sudden death. Therefore, we determined the effects of methadone and diazepam singly and in combination on cardiac action potentials (APs) and on the major ion channels responsible for cardiac repolarization. Using patch clamp recording in human stem cell-derived cardiomyocytes and stably transfected mammalian cells, we found that methadone produced concentration-dependent AP prolongation and ion channel block at low micromolar concentrations: hERG (IC50 = 1.7 µM), hNav1.5 (11.2 µM tonic block; 5.5 µM phasic block), and hCav1.2 (26.7 µM tonic block; 7.7 µM phasic block). Methadone was less potent in hKv4.3/hKChIP2.2 (IC50 = 39.0 µM) and hKvLQT1/hminK (53.3 µM). In contrast, diazepam blocked channels only at much higher concentrations and had no effect on AP duration at 1 µM. However, coadministration of 1-µM diazepam with methadone caused a statistically significant increase in AP duration and a 4-fold attenuation of hNav1.5 block (IC50 values were 44.2 µM and 26.6 µM, respectively, for tonic and phasic block), with no significant effect on methadone-induced block of hERG, hCav1.2, hKv4.3/hKChIP2.2, and hKvLQT1/hminK channels. Thus, although diazepam alone does not prolong the QT interval, the relief of methadone-induced Na channel block may leave hERG K channel block uncompensated, thereby increasing cardiac risk.

The action potential and comparative pharmacology of stem cell-derived human cardiomyocytes.

INTRODUCTION: The cardiac action potential (CAP) of stem cell-derived human cardiomyocytes (SC-hCMs) is potentially the most powerful preclinical biomarker for cardiac safety and efficacy in humans. Our experiments tested this hypothesis by examining the CAP and relevant pharmacology of these cells. METHODS: The electrophysiological and pharmacological profiles of SC-hCMs were compared to rabbit and canine Purkinje fibers (PFs). Ventricular SC-hCMs provided the dominant electrophysiological phenotype (approximately 82%) in a population of ventricular, atrial and nodal cardiomyocytes (CMs). The effects of reference compounds were measured in SC-hCMs using perforated patch, current clamp recording. Selective inhibitors of I(Kr), I(Ks), I(Ca,L), and I(Na), and norepinephrine (NE), were tested on SC-hCM action potentials (APs). RESULTS: AP prolongation was observed upon exposure to hERG channel blockers (terfenadine, quinidine, cisapride, sotalol, E-4031 and verapamil), with significantly shorter latencies than in PF assays. For the torsadogenic compounds, terfenadine and quinidine, SC-hCM AP prolongation occurred at significantly lower concentrations than in canine or rabbit PF APs. Moreover, the I(Ks) blocker chromanol 293B prolonged APs from SC-hCMs, whereas both rabbit and canine PF assays are insensitive to I(Ks) blockers in the absence of adrenergic preconditioning. Early afterdepolarizations (EADs) were induced by 100 nM E-4031 and 100 nM cisapride in the SC-hCM assay, but not in the canine or rabbit PF assay. Selective inhibition of I(Na) and I(Ca,L) slowed V(max) and shortened AP duration, respectively. NE prolonged the AP duration of SC-hCMs. DISCUSSION: The CAP of SC-hCMs has been validated as a powerful preclinical biomarker for cardiac safety and efficacy. In addition to its human nature, the SC-hCM AP assay removes diffusion delays, reduces test compound consumption, demonstrates an overall pharmacological sensitivity that is greater than conventional rabbit or canine PF assays, and accurately predicts cardiac risk of known torsadogenic compounds.

Evidence for a deep pore activation gate in small conductance Ca2+-activated K+ channels.

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (K(v)) channels, but are distinct in that they are gated solely by calcium (Ca(2+)), not voltage. For K(v) channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd(2+)) and barium (Ba(2+)), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd(2+) applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd(2+) and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba(2+) applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba(2+) is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba(2+) pre-block slows MTSEA modification of A384C in open but not in closed (Ba(2+)-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.

Kinetic relationship between the voltage sensor and the activation gate in spHCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarizations that cause an inward movement of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. The mechanism of how the motion of S4 charges triggers channel opening is unknown. Here, we used voltage clamp fluorometry (VCF) to detect S4 conformational changes and to correlate these to the different activation steps in spHCN channels. We show that S4 undergoes two distinct conformational changes during voltage activation. Analysis of the fluorescence signals suggests that the N-terminal region of S4 undergoes conformational changes during a previously characterized mode shift in HCN channel voltage dependence, while a more C-terminal region undergoes an additional conformational change during gating charge movements. We fit our fluorescence and ionic current data to a previously proposed 10-state allosteric model for HCN channels. Our results are not compatible with a fast S4 motion and rate-limiting channel opening. Instead, our data and modeling suggest that spHCN channels open after only two S4s have moved and that S4 motion is rate limiting during voltage activation of spHCN channels.

Slow conformational changes of the voltage sensor during the mode shift in hyperpolarization-activated cyclic-nucleotide-gated channels.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are activated by hyperpolarizations that cause inward movements of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. If HCN channels are held open for prolonged times (>50 ms), HCN channels undergo a mode shift, which in sea urchin (spHCN) channels induces a >50 mV shift in the midpoint of activation. The mechanism underlying the mode shift is unknown. The mode shift could be attributable to conformational changes in the pore domain that stabilize the open state of the channel, which would indirectly shift the voltage dependence of the channel, or attributable to conformational changes in the voltage-sensing domain that stabilize the inward position of S4, thereby directly shifting the voltage dependence of the channel. We used voltage-clamp fluorometry to detect S4 movements and to correlate S4 movements to the different activation steps in spHCN channels. We here show that fluorophores attached to S4 report on fluorescence changes during the mode shift, demonstrating that the mode shift is not simply attributable to a stabilization of the pore domain but that S4 undergoes conformational changes during the mode shift. We propose a model in which the mode shift is attributable to a slow, lateral movement in S4 that is triggered by the initial S4 gating-charge movement and channel opening. The mode shift gives rise to a short-term, activity-dependent memory in HCN channels, which has been shown previously to be important for the stable rhythmic firing of pacemaking neurons and could significantly affect synaptic integration.

Small conductance Ca2+-activated K+ channels and calmodulin: cell surface expression and gating.

Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming alpha subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.

Localization of the activation gate for small conductance Ca2+-activated K+ channels.

Small conductance Ca2+-activated K+ (SK) channels open in response to increased cytosolic Ca2+ and contribute to the afterhyperpolarization in many excitable cell types. Opening of SK channels is initiated by Ca2+ binding to calmodulin that is bound to the C terminus of the channel. Based on structural information, a chemomechanical gating model has been proposed in which the chemical energy derived from Ca2+ binding is transduced into a mechanical force that restructures the protein to allow K+ ion conduction through the pore. However, the residues that comprise the physical gate of the SK channels have not been identified. In voltage-gated K+ (Kv) channels, access to the inner vestibule is controlled by a bundle crossing formed by the intracellular end of the sixth transmembrane domain (S6) of each of the four channel subunits. Probing SK channels with internally applied quaternary amines suggests that the inner vestibules of Kv and SK channels share structural similarity. Using substituted cysteine accessibility mutagenesis, the relatively large molecule [2-(trimethylammonium)] methanethiosulfonate accessed positions near the putative bundle crossing more rapidly in the open than the closed state but did not modify S6 positions closer to the selectivity filter. In contrast, the smaller compound, 2-(aminoethyl) methanethiosulfonate (MTSEA), modified a position predicted to lie in the lumen immediately intracellular to the selectivity filter equivalently in the open and closed states. The pore blocker tetrabutylammonium impeded MTSEA access to this position in both open and closed channels. The results suggest that the SK channel gate is not formed by the cytoplasmic end of S6 but resides deep in the channel pore in or near the selectivity filter.