p.D1690N Nav1.5 rescues p.G1748D mutation gating defects in a compound heterozygous Brugada syndrome patient.

BACKGROUND: We identified 2 compound heterozygous mutations (p.D1690N and p.G1748D) in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) in a proband diagnosed with Brugada syndrome type 1. Furthermore, in the allele encoding the p.D1690N mutation, the p.H558R polymorphism was also detected. OBJECTIVE: The purpose of this study was to analyze the functional properties of the mutated channels as well as the putative modulator effects produced by the presence of the polymorphism. METHODS: Wild-type and mutated human Nav1.5 channels were expressed in Chinese hamster ovary cells and recorded using whole-cell patch-clamp technique. RESULTS: Separately, both p.D1690N and p.G1748D mutations produced a marked reduction in peak Na(+) current density (>80%), mainly due to their limited trafficking toward the membrane. Furthermore, p.G1748D mutation profoundly affected channel gating. Both p.D1690N and p.G1748D produced a marked dominant negative effect when cotransfected with either wild-type or p.H558R channels. Conversely, p.H558R was able to rescue defective trafficking of p.D1690N channels toward the membrane when both polymorphism and mutation were in the same construct. Surprisingly, cotransfection with p.D1690N, either alone or together with the polymorphism (p.H558R-D1690N), completely restored the profound gating defects exhibited by p.G1748D channels but only slightly rescued their trafficking. CONCLUSIONS: Our results add further support to the hypothesis that Nav1.5 subunits interact among them before trafficking toward the membrane and suggest that a missense mutation can "rescue" the defective gating produced by another missense mutation.

Propafenone blocks human cardiac Kir2.x channels by decreasing the negative electrostatic charge in the cytoplasmic pore.

Human cardiac inward rectifier current (IK1) is generated by Kir2.x channels. Inhibition of IK1 could offer a useful antiarrhythmic strategy against fibrillatory arrhythmias. Therefore, elucidation of Kir2.x channels pharmacology, which still remains elusive, is mandatory. We characterized the electrophysiological and molecular basis of the inhibition produced by the antiarrhythmic propafenone of the current generated by Kir2.x channels (IKir2.x) and the IK1 recorded in human atrial myocytes. Wild type and mutated human Kir2.x channels were transiently transfected in CHO and HEK-293 cells. Macroscopic and single-channel currents were recorded using the patch-clamp technique. At concentrations >1µM propafenone inhibited IKir2.x the order of potency being Kir2.3∼IK1>Kir2.2>Kir2.1 channels. Blockade was irrespective of the extracellular K(+) concentration whereas markedly increased when the intracellular K(+) concentration was decreased. Propafenone decreased inward rectification since at potentials positive to the K(+) equilibrium potential propafenone-induced block decreased in a voltage-dependent manner. Importantly, propafenone favored the occurrence of subconductance levels in Kir2.x channels and decreased phosphatidylinositol 4,5-bisphosphate (PIP2)-channel affinity. Blind docking and site-directed mutagenesis experiments demonstrated that propafenone bound Kir2.x channels at the cytoplasmic domain, close to, but not in the pore itself, the binding site involving two conserved Arg residues (residues 228 and 260 in Kir2.1). Our results suggested that propafenone incorporated into the cytoplasmic domain of the channel in such a way that it decreased the net negative charge sensed by K(+) ions and polyamines which, in turn, promotes the appearance of subconductance levels and the decrease of PIP2 affinity of the channels.

Functional effects of a missense mutation in HERG associated with type 2 long QT syndrome.

BACKGROUND: Long QT syndrome (LQTS) is characterized by a prolonged QT interval that can lead to severe ventricular arrhythmias (torsades de pointes) and sudden death. Congenital LQTS type 2 (LQT2) is due to loss-of-function mutations in the KCNH2 gene encoding Kv11.1 channels responsible for the rapid component of the delayed rectifier current. OBJECTIVE: The purpose of this study was to determine the functional properties of the LQT2-associated mutation p.E637G found in a Spanish family. METHODS: Wild-type (WT) and p.E637G Kv11.1 channels were transiently transfected in Chinese hamster ovary cells, and currents were recorded using the patch-clamp technique. RESULTS: The p.E637G channels lost inward rectification and K(+) selectivity, generating small but measurable slowly activating, noninactivating currents. These important alterations were corrected neither by cotransfection with WT channels nor by incubation at low temperatures or with pharmacological chaperones. As a consequence of its effects on channel gating, the mutation significantly reduced the outward repolarizing current during the action potential (AP), resulting in a marked lengthening of the duration of a simulated human ventricular AP. CONCLUSION: We have identified and characterized an LQT2-associated mutation that through removal of C-type inactivation and reduction of K(+) selectivity causes the QT prolongation observed in the patients carrying the mutation. Moreover, the results obtained demonstrate the importance of the glutamic acid at position 637 for the inactivation process and K(+) selectivity of Kv11.1 channels.

In humans, chronic atrial fibrillation decreases the transient outward current and ultrarapid component of the delayed rectifier current differentially on each atria and increases the slow component of the delayed rectifier current in both.

OBJECTIVES: The purpose of this study was to compare the voltage-dependent K(+) currents of human cells of the right and left atria and determine whether electrical remodeling produced by chronic atrial fibrillation (CAF) is chamber-specific. BACKGROUND: Several data point to the existence of interatrial differences in the repolarizing currents. Therefore, it could be possible that CAF-induced electrical remodeling differentially affects voltage-dependent K(+) currents in each atrium. METHODS: Currents were recorded using the whole-cell patch-clamp in myocytes from left (LAA) and right atrial appendages (RAA) obtained from sinus rhythm (SR) and CAF patients. RESULTS: In SR, LAA and RAA myocytes were divided in 3 types, according to their main voltage-dependent repolarizing K(+) current. CAF differentially modified the proportion of these 3 types of cells on each atrium. CAF reduced the Ca(2+)-independent 4-aminopyridine-sensitive component of the transient outward current (I(to1)) more markedly in the LAA than in the RAA. Therefore, an atrial right-to-left I(to1) gradient was created by CAF. In contrast, the ultrarapid component of the delayed rectifier current (I(Kur)) was more markedly reduced in the RAA than in the LAA, thus abolishing the atrial right-to-left I(Kur) gradient observed in SR. Importantly, in both atria, CAF increased the slow component of the delayed rectifier current (I(Ks)). CONCLUSIONS: Our results demonstrated that in SR there are intra-atrial heterogeneities in the repolarizing currents. CAF decreases I(to1) and I(Kur) differentially in each atrium and increases I(Ks) in both atria, an effect that further promotes re-entry.