Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K + current (I Kr ) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix, and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K + current (I Kr ) and are essential for regulating the duration of the plateau phase of the cardiac action potential 1,2 . Inherited loss-of-function mutations in hERG1 can lead to life threatening torsades de pointes (TdP) arrhythmia 3 , while gain-of-function mutations are associated with short QT syndrome 4 . Most frequently, TdP arrhythmia is an acquired disorder, resulting from "off-target" block of this channel by structurally diverse drugs including antiarrhythmics, antihistamines, antipsychotics and antibiotics 5 . Since this inhibition can lead to sudden cardiac death, several pharmaceuticals such as cisapride or terfenadine were withdrawn from the market, or had their use severely restricted 6,7 . Recently, the Cardiac Safety Research Consortium (CSRC) and the Food and Drug Administration (FDA) proposed a new cardiac safety paradigm labelled "Comprehensive In Vitro Proarrhythmia Assay" (CiPA). The new CiPA guidelines emphasize the importance of studying pharmacological effects of drugs on three different ion channel types including hERG, Nav1.5 and Cav1.2, which proposed to play an important role in shaping the ventricular action potential 8 . hERG1 blockers might also have beneficial therapeutic potential. During routine preclinical screening for hERG1, new modulators, so-called activators, have been identified. These modulators may have the potential of shortening the action potential duration 9 . Thus, they might be beneficial for the treatment of inherited long QT syndrome.Great efforts have been directed toward a better understanding of the molecular and structural mechanisms of hERG1 channel drug interactions, including in vivo, in vitro and in silico approaches (for a recent reviews see Durdagi, S. et al. 10 and Vandenberg, J. et al. 11 ). Substantial progress has been made by identifying amino acids essential for drug block. The majority of hERG inhibitors are interacting with the pore module. This homo tetrameric module consists of an outer S5 helix, a...

PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.

Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of Ca V 1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4 N ), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4 N channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments ("GAGA ring": G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4 N was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6-IVS6, which also affect activation gating, were not rescued by IIS4 N . Thus, the rescue of gating distortions in segments IS6-IVS6 by IIS4 N is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.

BACKGROUND AND PURPOSEDrug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. EXPERIMENTAL APPROACHhERG and KIR2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr. Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. KEY RESULTSStructural modifications of pentamidine differentially affected plasma membrane levels of hERG and KIR2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not KIR2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. CONCLUSIONS AND IMPLICATIONSDrug-induced trafficking defects can be minimized if certain chemical features are avoided or 'synthesized out'; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS. AbbreviationsER, endoplasmic reticulum; hERG, human ether-a-go-go-related gene; IK1, cardiac inward rectifying K + current; IKr, rapid component of the delayed rectifier K

I. M. LINDER AND D. M. J. QUASTEL of a single species of membrane channel that allows the free movement of K+, Na+, NH+, and Li+ ions along their electrochemical gradients. The channel discriminates little between these ions. The apparent order of permeability is Li+ > NH + > Na+ 3 K+. The apparent permeability per channel corresponds to that expected for channels of about 6-4 A diameter, 100 A length, and ionic mobility the same as in dilute solution.