Abstract-The N629D mutation, adjacent to the GFG signature sequence of the HERG1 A K ϩ channel, causes long-QT syndrome (LQTS). Expression of N629D in Xenopus oocytes produces a rapidly activating, noninactivating current. N629D is nonselective among monovalent cations; permeation of K ϩ was similar to that of Na ϩ or Cs ϩ . During repolarization to potentials between Ϫ30 and Ϫ70 mV, N629D manifested an inward tail current, which was abolished by replacement of extracellular Na ϩ (Na ϩ e ) with extracellular N-methyl-D-glucamine (NMG e ). Because LQTS occurs in heterozygous patients, we coexpressed N629D and wild type (WT) at equimolar concentrations. Heteromultimer formation was demonstrated by analyzing the response to 0 [K ϩ ] e . The outward time-dependent current was nearly eliminated for WT at 0 [K ϩ ] e , whereas no reduction was observed for homomultimeric N629D or for the equimolar coexpressed current. To assess physiological significance, dofetilide-sensitive currents were recorded during application of simulated action potential clamps. During phase 3 repolarization, WT manifested outward currents, whereas homomultimeric N629D manifested inward depolarizing currents. During coexpression studies, variable phenotypes were observed ranging from a reduction in outward repolarizing current to net inward depolarizing current during phase 3. In summary, N629D replaces the WT outward repolarizing tail current with an inward depolarizing sodium current, which is expected to delay later stages of repolarization and contribute to arrhythmogenesis. Thus, the consequences of N629D resemble the pathophysiology seen in LQT3 Na ϩ channel mutations and may be considered the first LQTS K Key Words: long-QT syndrome Ⅲ HERG1 Ⅲ K ϩ channel Ⅲ gain of function F amilial long-QT syndrome (LQTS) results from defects in sodium and potassium ion channels that cause prolongation of cardiac repolarization and arrhythmias. 1 LQT2 is associated with mutations of the human ether-a-go-go-related gene, HERG1. [2][3][4][5][6][7][8][9][10] The HERG1 primary transcript is alternatively processed, giving rise to at least 3 functional mRNAs, HERG1 A, HERG1 AЈ, and HERG1 B, encoding proteins with distinct physiological properties. 11,12 There are several mechanisms by which individual mutations in HERG1 produce LQTS. 5-9 Some exert a dominant phenotype through loss of repolarizing current. These include mutations that either cause defects in intracellular transport or result in channels that do not open. Alternatively, V630L forms heterotetramers with wild type (WT) that have reduced open probability due to a negative shift in voltage dependence of inactivation. Abnormally fast deactivation mutations caused by mutations in the N terminus of HERG1 A result in reduction of outward current during phase 3 repolarization and thus LQTS. 10 The recently described N629D mutation 9 is of particular interest, as it alters the pore selectivity signature sequence from GFGN to GFGD. 13 In most K ϩ channels, including the extensively studied Shaker chann...

Abstract-Loss-of-function mutations in the human ERG1 potassium channel (hERG1) frequently underlie the long QT2 (LQT2) syndrome. The role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous, loss-of-function LQT2 syndrome mutation. The hERG N629D mutation was introduced into the orthologous mouse gene, mERG, by homologous recombination in mouse embryonic stem cells. Intact homozygous embryos showed abrupt cessation of the heart beat. N629D/N629D embryos die in utero by embryonic day 11.5. Their developmental defects include altered looping architecture, poorly developed bulbus cordis, and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of I Kr function, depolarized resting potential, prolonged action potential duration (LQT), failure to repolarize, and propensity to oscillatory arrhythmias. N629D/N629D myocytes manifest calcium oscillations and increased sarcoplasmic reticulum Ca ϩ2 content. Although the N629D/N629D protein is synthesized, it is mainly located intracellularly, whereas ϩ/ϩ mERG protein is mainly in plasmalemma. N629D/N629D embryos show robust apoptosis in craniofacial regions, particularly in the first branchial arch and, to a lesser extent, in the cardiac outflow tract. Because deletion of Hand2 produces apoptosis, in similar regions and with a similar final developmental phenotype, Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. In conclusion, loss of I Kr function in N629D/N629D cardiovascular system leads to defects in cardiac ontogeny in the first branchial arch, outflow tract, and the right ventricle. Key Words: KCNH2 (hERG) Ⅲ knock-in mouse Ⅲ embryo developmental defect T he human ERG gene (hERG/KCNH2) encodes a potassium channel that is important in the late stage of action potential repolarization in heart. Mutations in this gene, which generally reduce plasmalemmal expression of hERG, lead to the long QT2 (LQT2) syndrome in humans. [1][2] Patients with the LQT2 syndrome have a delay in cardiac repolarization that predisposes them to cardiac arrhythmias that can be lethal. 1,2 Mutations in hERG are associated with embryonic lethality and the sudden infant death syndrome. [3][4] Although the LQT2 syndrome generally occurs in individuals heterozygous for the mutant allele, individuals homozygous for the exon 4 duplication manifest embryonic lethality or are rescued in the neonatal period by pacing. 5 Although not widely recognized, mutations of' hERG appear to be associated with structural congenital cardiovascular anomalies including: tetralogy of Fallot, atrial-septal defects, ventricularseptal defects, and patent ductus arteriosus. 6 -9 Mouse ERG (mERG) is the dominant repolarizing current in the mouse embryonic heart. 10 A channel analogous to hERG is expressed in differentiating quail neural crest cells 11 early in development. These data imply a potential role of the ERG potass...

To test the hypothesis that variations in cerebrovascular reactivity to 5-HT among arteries of different size or type, during maturation, or during acclimatization to high altitude involve differences in serotonergic receptor subtype, we determined relative agonist potency orders and antagonist affinities in common carotid (Com), main branch middle cerebral (Main), and second branch middle cerebral (2BR) arteries from term fetal lambs and nonpregnant adult sheep acclimatized at sea level or at an altitude of 3,820 m for ≈110 days. In normoxic adult Com segments, agonist potency order was 5-hydroxytryptamine (5-HT) > 5-carboxamidotryptamine (5-CT) ≥ 8-hydroxy-2(di- n-propylamino)tetraline (8-OH-DPAT); sumatriptan (Suma) produced no contractile response; and antagonist dissociation constant (pKb) values were 9.4 and 9.5 for ketanserin against 5-HT and 5-CT, 7.5 for GR-127935 against 5-HT, and 7.2 for SB-206553 against 5-HT. In normoxic adult Main segments, agonist potency order was 5-HT > 5-CT ≥ Suma ≥ DPAT, and pKb values were 9.1 and 9.2 for ketanserin against 5-HT and 5-CT and 7.4 and 8.5 for GR-127935 against 5-HT and Suma, respectively. In the 2BR segments from normoxic adults, agonist potency order was 5-CT > 5-HT > Suma > DPAT and pKb values were 7.4 and 7.2 for ketanserin against 5-HT and 5-CT and 10.0 and 8.7 for GR-127935 against 5-HT and Suma, respectively. Compared with normoxic adults, none of these values were significantly different in hypoxic adults and in fetuses only the pKb values for ketanserin against 5-HT in the 2BR segments (8.8) were greater. From these results we propose that the ratio of 5-HT2 to 5-HT1 receptors is greatest in the Com and decreases progressively to its smallest values in 2BR or smaller segments. Because this gradient appears stable and relatively resistant to the effects of maturation and chronic hypoxia, changes in reactivity associated with these perturbations may involve alterations in receptor density and/or coupling efficiency for 5-HT in ovine cranial arteries.

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This study reports a novel therapeutic strategy and mechanism to partially restore physiologic function in this HERG LQTS mutation.

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Responses to hypoxia of lung arteries (200-350 microns) from control (C) and chronically hypoxic (CH) rats were compared in a myograph before and after blockade of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME). After precontraction with prostaglandin F2 alpha (PGF2 alpha), hypoxia caused a four-phase tension change: brief dilation, transient contraction, prolonged dilation, and slow contraction (we studied the first three phases). In CH rats, the first dilation and first contraction were significantly reduced. After L-NAME, the first dilation was reduced in C rats and abolished in CH rats; thus the first phase is attributable to NO release and is affected by chronic hypoxia. The first contractile phase was significantly reduced by L-NAME in C but not in CH rats, where it was small. Thus NO synthase inhibition inhibits hypoxic constriction in isolated vessels, whereas it enhances hypoxic constriction in perfused lungs. The third dilator phase was unaffected by chronic hypoxia; it was increased after L-NAME in CH rats. Thus, in vitro, responses to hypoxia are complex; there is a balance between two dilator and two constrictor processes.