Uncoupling of Gating Charge Movement and Closure of the Ion Pore During Recovery from Inactivation in the Kv1.5 Channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na+ in their inactivated states when K+ is absent. Replacement of K+ with Na+ or NMG+ allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at −100 mV was 11.1 ± 0.4 ms (n = 10) and increased to 19.0 ± 1.6 ms (n = 3) when NMG+o was replaced by Na+o. However, the decay of the Na+ tail currents through inactivated channels at −100 mV had a time constant of 129 ± 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na+ tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na+-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na+ but not symmetrical Cs+. This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether

NH2-terminal Inactivation Peptide Binding to C-type–inactivated Kv Channels

In many voltage-gated K+ channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na+ permeability of C-type–inactivated K+ channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type–inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na+ tail currents normally observed through C-type–inactivated channels, an effective blockade of the peak Na+ tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type–inactivated channels. In C-type–deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type–inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na+ tail of C-type–inactivated channels. Stable binding between the inactivation peptide and the C-type–inactivated state results in slower current decay, and a reduction of the Na+ tail current magnitude, due to slower transition of channels through the Na+-permeable states traversed during recovery from inactivation

Gating current studies reveal both intra- and extracellular cation modulation of K+ channel deactivation

The presence of permeant ions can modulate the rate of gating charge return in wild-type human heart K+ (hKv1.5) channels. Here we employ gating current measurements in a non-conducting mutant, W472F, of the hKv1.5 channel to investigate how different cations can modulate charge return and whether the actions can be specifically localized at the internal as well as the external mouth of the channel pore.Intracellular cations were effective at accelerating charge return in the sequence Cs+ > Rb+ > K+ > Na+ > NMG+. Extracellular cations accelerated charge return with the selectivity sequence Cs+ > Rb+ > Na+ = NMG+.Intracellular and extracellular cation actions were of relatively low affinity. The Kd for preventing slowing of the time constant of the off-gating current decay (τoff) was 20.2 mM for intracellular Cs+ (Csi+) and 358 mM for extracellular Cs+ (Cso+).Both intracellular and extracellular cations can regulate the rate of charge return during deactivation of hKv1.5, but intracellular cations are more effective. We suggest that ion crystal radius is an important determinant of this action, with larger ions preventing slowing more effectively. Important parallels exist with cation-dependent modulation of slow inactivation of ionic currents in this channel. However, further experiments are required to understand the exact relationship between acceleration of charge return and the slowing of inactivation of ionic currents by cations

Blocking eukaryotic initiation factor 4F complex formation does not inhibit the mTORC1-dependent activation of protein synthesis in cardiomyocytes

Activation of the mammalian target of rapamycin complex 1 (mTORC1) causes the dissociation of eukaryotic initiation factor 4E complex (eIF4E)-binding protein 1 (4E-BP1) from eIF4E, leading to increased eIF4F complex formation. mTORC1 positively regulates protein synthesis and is implicated in several diseases including cardiac hypertrophy, a potentially fatal disorder involving increased cardiomyocyte size. The importance of 4E-BP1 in mTORC1-regulated protein synthesis was investigated by overexpressing 4E-BP1, which blocks eIF4F formation in isolated primary cardiomyocytes without affecting other targets for mTORC1 signaling. Interestingly, blocking eIF4F formation did not impair the degree of activation of overall protein synthesis by the hypertrophic agent phenylephrine (PE), which, furthermore, remained dependent on mTORC1. Overexpressing 4E-BP1 also only had a small effect on PE-induced cardiomyocyte growth. Overexpressing 4E-BP1 did diminish the PE-stimulated synthesis of luciferase encoded by structured mRNAs, confirming that such mRNAs do require eIF4F for their translation in cardiomyocytes. These data imply that the substantial inhibition of cardiomyocyte protein synthesis and growth caused by inhibiting mTORC1 cannot be attributed to the activation of 4E-BP1 or loss of eIF4F complexes. Our data indicate that increased eIF4F formation plays, at most, only a minor role in the mTORC1-dependent activation of overall protein synthesis in these primary cells but is required for the translation of structured mRNAs. Therefore, other mTORC1 targets are more important in the inhibition by rapamycin of the rapid activation of protein synthesis and of cell growth. <br/

De Novo Mutation in the SCN5A Gene Associated with Brugada Syndrome

Background: Brugada syndrome (BrS) is a genetically determined cardiac electrical disorder, characterized by typical electrocardiography (ECG) alterations, and it is an arrhythmogenic syndrome that may lead to sudden cardiac death. The most common genotype found among BrS patients is caused by mutations in the SCN5A gene, which lead to a loss of function of the cardiac sodium (Na+) channel (Nav1.5) by different mechanisms. Methods: The assay of confocal laser microscopy and western blot were used to identify the expression and location of L812Q at the cell surface. Characterization of Nav1.5 L812Q mutant Na+ channels was text by patch-clamp recordings, and the PHYRE2 server was used to build a model for human Nav1.5 channel. Results: Here, we report that a novel missense SCN5A mutation, L812Q, localized in the DII-S4 transmembrane region of the Nav1.5 channel protein, was identified in an index patient who showed a typical BrS type-1 ECG phenotype. The mutation was absent in the patient's parents and brother. Heterologous expression of the wild-type (WT) and L812Q mutant Nav1.5 channels in human embryonic kidney cells (HEK293 cells) reveals that the mutation results in a reduction of Na+ current density as well as ∼20 mV hyperpolarizing shift of the voltage dependence of inactivation. The voltage dependence of activation and the time course for recovery from inactivation are not affected by the mutation. The hyperpolarizing shift of the voltage dependence of inactivation caused a reduction of the Na+ window current as well. In addition, western blot and confocal laser microscopy imaging experiments showed that the mutation causes fewer channel to be expressed at the membrane than WT channel. A large proportion of the mutant channels are retained in the cytoplasm, probably in the endoplasmic reticulum. Conclusion: The decrease of channel expression, hyperpolarizing shift of voltage dependence of inactivation, and a decline of Na+ window current caused by L812Q mutation lead to a reduction of Na+ current during the upstroke and the repolarization phases of cardiac action potential, which contribute to the development of BrS