Properties of WT and mutant hERG K(+) channels expressed in neonatal mouse cardiomyocytes.

Section of Cardiovascular Medicine, Departments of Medicine and Physiology, University of Wisconsin, Madison, Wisconsin, USA.
AJP Heart and Circulatory Physiology (Impact Factor: 4.01). 04/2010; 298(6):H1842-9. DOI: 10.1152/ajpheart.01236.2009
Source: PubMed

ABSTRACT Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming alpha-subunits that coassemble to form rapidly activating delayed rectifier K(+) current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The DeltaY475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue.

  • [Show abstract] [Hide abstract]
    ABSTRACT: In this communication, we present a universal read-out system, which can be used to decode polarimetric fiber-optic sensors based on highly birefringent fibers. All such sensors use the same sensing principle, relying upon the dependence of modal birefringence on different physical parameters. To register the measurand-induced phase changes between polarization modes, we use the coherence-addressing principle. This requires that the interrogated sensor be powered by a broadband source (superluminescent diode) and that the total optical path delay introduced by the sensor be balanced in the decoding interferometer. The system performance in decoding temperature, elongation and hydrostatic pressure sensor is demonstrated.
    01/2002; 2.
  • [Show abstract] [Hide abstract]
    ABSTRACT: KCNH2 encodes the Kv11.1 channel, which conducts the rapidly activating delayed rectifier K(+) current (I Kr) in the heart. KCNH2 mutations cause type 2 long QT syndrome (LQT2), which increases the risk for life-threatening ventricular arrhythmias. LQT2 mutations are predicted to prolong the cardiac action potential (AP) by reducing I Kr during repolarization. Kv11.1 contains several conserved basic amino acids in the fourth transmembrane segment (S4) of the voltage sensor that are important for normal channel trafficking and gating. This study sought to determine the mechanism(s) by which LQT2 mutations at conserved arginine residues in S4 (R531Q, R531W or R534L) alter Kv11.1 function. Western blot analyses of HEK293 cells transiently expressing R531Q, R531W or R534L suggested that only R534L inhibited Kv11.1 trafficking. Voltage-clamping experiments showed that R531Q or R531W dramatically altered Kv11.1 current (I Kv11.1) activation, inactivation, recovery from inactivation and deactivation. Coexpression of wild type (to mimic the patients' genotypes) mostly corrected the changes in I Kv11.1 activation and inactivation, but deactivation kinetics were still faster. Computational simulations using a human ventricular AP model showed that accelerating deactivation rates was sufficient to prolong the AP, but these effects were minimal compared to simply reducing I Kr. These are the first data to demonstrate that coexpressing wild type can correct activation and inactivation dysfunction caused by mutations at a critical voltage-sensing residue in Kv11.1. We conclude that some Kv11.1 mutations might accelerate deactivation to cause LQT2 but that the ventricular AP duration is much more sensitive to mutations that decrease I Kr. This likely explains why most LQT2 mutations are nonsense or trafficking-deficient.
    Journal of Membrane Biology 04/2013; · 2.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: Type 2 long QT syndrome (LQT2) involves mutations in the human ether a-go-go-related gene (hERG or KCNH2). T421M, a S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking and pharmacological mechanisms in adult rat ventricular myocytes (ARVMs). METHODS AND RESULTS: Isolated ARVMs were infected with WT- and T421M-Kv11.1 expressing adenovirus and analyzed using patch clamp, Western blot and confocal imaging techniques. Expression of WT- or T421M-Kv11.1 produced peak tail current (I(Kv11.1)) of 8.78±1.18 and 1.91±0.22 pA/pF, respectively. Loss of mutant I(Kv11.1) resulted from, 1) a partially trafficking-deficient channel protein with reduced cell surface expression, and 2) altered channel gating with a positive shift in the voltage-dependence of activation and altered kinetics of activation and deactivation. Co-expression of WT+T421M-Kv11.1 resulted in heterotetrameric channels that remained partially trafficking-deficient with only a minimal increase in peak I(Kv11.1) density, whereas the voltage-dependence of channel gating became "WT-like". In the ARVM model, both WT- and T421M-Kv11.1 channels responded to β-adrenergic stimulation by increasing I(Kv11.1). CONCLUSIONS: The T421M-Kv11.1 mutation caused a loss of I(Kv11.1) through interactions of abnormal protein trafficking and channel gating. Furthermore, for co-expressed WT+T421M-Kv11.1 channels, different dominant negative interactions govern protein trafficking and ion channel gating, and these are likely to be reflected in the clinical phenotype. Our results also show that WT and mutant Kv11.1 channels responded to β-adrenergic stimulation.
    Circulation 11/2012; · 14.95 Impact Factor