Inward rectifier K+ channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg2+ and a Mg(2+)-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K+ channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.
This is the first study to demonstrate differential responses of LQTS patients to interventions targeted to their specific genetic defect. These findings also suggest that LQT3 patients may be more likely to benefit from Na+ channel blockers and from cardiac pacing because they would be at higher risk of arrhythmia at slow heart rates. Conversely, LQT2 patients may be at higher risk to develop syncope under stressful conditions because of the combined arrhythmogenic effect of catecholamines with the insufficient adaptation of their QT interval when heart rate increases.
Abstract-The human ether-a-gogo-related gene (hERG) encodes the ␣ subunit of the cardiac potassium current I Kr .Several mutations in hERG produce trafficking-deficient channels that may cause hereditary long-QT syndrome and sudden cardiac death. Although hERG currents have been studied extensively, little is known about the proteins involved in maturation and trafficking of hERG. Using immunoprecipitations, we show that the cytosolic chaperones heat shock protein (Hsp) 70 and Hsp90, but not Grp94, interact with hERG wild type (WT) during maturation. The specific Hsp90 inhibitor geldanamycin prevents maturation and increases proteasomal degradation of hERG WT, while reducing hERG currents in heterologous expression systems. In ventricular myocytes, inhibition of Hsp90 also decreases I Kr , whereas geldanamycin had no effect on I Ks or heterologously expressed Kv2.1 and Kv1.5 currents. Both Hsp90 and Hsp70 interact directly with the core-glycosylated form of hERG WT present in the endoplasmic reticulum but not the fully glycosylated, cell-surface form. For the trafficking-deficient LQT2 mutants, hERG R752W and hERG G601S, interactions with Hsp90 and Hsp70 are increased as both mutants remained tightly associated with Hsp90 and Hsp70 in the endoplasmic reticulum. Incubation at lower temperature for R752W or with the hERG blocker astemizole for G601S dissociates channel-chaperone complexes and restores trafficking. In contrast, nonfunctional but traffickingcompetent hERG G628S is released from chaperone complexes during maturation comparable to WT. We conclude that
The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac delayed rectifier K+ current (IKr). HERG/IKr channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in Xenopus oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K+ channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.
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.
Voltage-activated potassium channels play an important part in the control of excitability in nerve and muscle. Different K+ channels are involved in establishing the resting potential, determining the duration of action potentials, modulation of transmitter release, and in rhythmic firing patterns and delayed excitation. Using in vitro transcripts made from a directional complementary DNA library we have isolated, by expression cloning in Xenopus oocytes, a novel K+-channel gene (drk1). Functionally, drk1 encodes channels that are K+ selective and belong to the delayed rectifier class of channels, rather than the A-type class encoded by the Shaker gene of Drosophila. The channels show sigmoidal voltage-dependent activation and do not inactivate within 500 ms. Structurally, drk1 encodes an amino-acid sequence which is more closely related to the Drosophila Shab gene than to the Shaker gene.
In human myocardium, the nature of the K+ currents mediating repolarization of the action potential is still speculative. Delayed rectifier channels have recently been cloned from human myocardium, but it is unclear whether or not these currents are involved in the termination of the cardiac action potential plateau. In intact human atrial myocytes, we have identified a rapid delayed rectifier K+ current with properties and kinetics identical to those expressed by a K+ channel clone (fHK) isolated from human heart and stably incorporated into a human cell line for the first time. The myocyte current amplitude was 3.6 +/- 0.2 pA/pF (at +20 mV, n = 15) and activated with a time constant of 13.1 +/- 2 milliseconds at 0 mV (n = 15). The half-activation potential (V0.5) was -6 +/- 2.5 mV (n = 10) with a slope factor (k) of 8.6 +/- 2.2 (n = 10). The heterologously expressed fHK current amplitude was 136 pA/pF (at +20 mV, n = 9) with an activation time constant of 11.8 +/- 4.6 milliseconds at 0 mV; V0.5 was 4.1 +/- 2.4 mV (mean +/- SEM, n = 8); and k was 7.0. The conductance of single fHK channels was 16.9 picosiemens in 5 mM bath K+. Both native and cloned channel currents inactivated partially during sustained depolarizing pulses. Both currents were blocked by micromolar concentrations of 4-aminopyridine and were relatively insensitive to tetraethylammonium ions and class III antiarrhythmic agents.(ABSTRACT TRUNCATED AT 250 WORDS)
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