Abstract-Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC 50 . The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS. (Circ Res. 2008;103:e81-e95.)Key Words: Kv11.1 Ⅲ KCNH2 Ⅲ ether-à-go-go Ⅲ arrhythmia Ⅲ potassium channels C ardiac I Kr is a potassium current contributing to ventricular repolarization in mammalian heart. 1,2 The molecular basis of cardiac I Kr was first elucidated when its unique biophysical and pharmacological properties were largely reproduced by heterologous expression of the hERG1 gene (human ether-à-go-go-related gene) (or KCNH2). 3,4 Together with the discovery of KCNH2 mutations as the pathogenic substrate in families with type 2 long-QT syndrome (LQTS), 5 these studies explained the underlying cause of disease as a loss of cardiac I Kr . They also identified hERG 1 channels as a molecular target for acquired LQTS, a much more prevalent form of the disease arising from I Kr block primarily by drugs intended for other therapeutic targets. 6 In either manifestation, LQTS is characterized by prolonged ventricular action potentials and a susceptibility to potentially life-threatening arrhythmias known as torsades de pointes (TdP). 7 Our understanding of how I Kr contributes to ventricular repolarization is based primarily on heterologous expression of the originally identified hERG 1a subunit. 3,4,8,9 Like other voltage-gated potassium channels, hERG 1a channels activate and inactivate on depolarization. However, b...

Defects in the trafficking of subunits encoded by the human ether-à-go-go-related gene (hERG1) can lead to catastrophic arrhythmias and sudden cardiac death due to a reduction in I Kr -mediated repolarization. Native I Kr channels are composed of two ␣ subunits, hERG 1a and 1b. In heterologous expression systems, hERG 1b subunits efficiently produce current only in heteromeric combination with hERG 1a. We used Western blot analysis and electrophysiological recordings in HEK-293 cells and Xenopus oocytes to monitor hERG 1b maturation in the secretory pathway and to determine the factors regulating surface expression of hERG 1b subunits. We found that 1b subunits expressed alone were largely retained in the endoplasmic reticulum (ER), thus accounting for the poor functional expression of homomeric 1b currents. Association with hERG 1a facilitated 1b ER export and surface expression. We show that hERG 1b subunits fail to mature because of an "RXR" ER retention signal specific to the 1b N terminus of the human sequence and not conserved in other species. Mutating the RXR facilitated maturation and functional expression of homomeric hERG 1b channels in a charge-dependent manner. Co-expression of the 1b RXR mutants with hERG 1a did not further enhance 1b maturation, suggesting that hERG 1a promotes 1b trafficking by overcoming the RXR-mediated retention. Thus, selective trafficking mechanisms regulate subunit composition of surface hERG channels.Voltage-gated potassium (K ϩ ) channels encoded by the human ether-à-go-go-related gene (hERG1 or KCNH2) mediate the repolarizing cardiac current I Kr (1, 2). Perturbation of I Kr due to mutations in the hERG1 gene or drug block of hERG 2 channels can cause sudden cardiac death associated with long QT syndrome (LQTS) (3).Native I Kr channels are composed of hERG 1a and 1b ␣ subunits encoded by alternate transcripts of the hERG1 gene (4). hERG 1a and 1b subunits have identical transmembrane and C-terminal sequences but divergent N termini (5, 6), which interact to promote heteromeric assembly early in channel biogenesis (7). When expressed heterologously, hERG 1a homomeric and 1a/1b heteromeric channels yield robust currents but with distinct properties because of the divergent N termini of the constituent subunits (5, 8, 9). However, hERG 1b homomers produce undetectable or very small currents (5). Why hERG 1b functional expression is inefficient, or how hERG 1a promotes 1b surface expression, is unknown.In this study we found that hERG 1b protein efficiently exited the ER only in the presence of hERG 1a. We tested the hypothesis that hERG 1b subunits possess ER retention/retrieval signals that are inactivated upon association with the 1a subunit, thus favoring surface expression of the heteromeric channel. Of two RXR motifs (two arginines separated by any single residue) in the 1b N terminus, surprisingly only the one motif specific to the human sequence and not conserved in other organisms subserved ER retention. Moreover, mutations within this signal promoted surface expres...

Galectin-3 (Gal-3), a member of the β-galactoside-binding protein family, is implicated in a wide variety of human diseases. Identification of Gal-3 inhibitors with the right combination of potency (against both human and mouse Gal-3) and pharmacokinetic properties to fully evaluate the potential of Gal-3 for therapeutic intervention has been a major challenge due to the characteristics of its binding pocket: high hydrophilicity and key structural differences between human Gal-3 and the mouse ortholog. We report the discovery of a novel series of monosaccharide-based, highly potent, and orally bioavailable inhibitors of human and mouse Gal-3. The novel monosaccharide derivatives proved to be selective for Gal-3, the only member of the chimeric type of galectins, over Gal-1 and Gal-9, representative of the prototype and tandem-repeat type of galectins, respectively. The proposed binding mode for the newly identified ligands was confirmed by an X-ray cocrystal structure of a representative analogue bound to Gal-3 protein.