2-(2-Furanyl)-7-phenyl[1,2,4]triazolo[1,5-c]pyrimidin-5-amine analogs as adenosine A2A antagonists: the successful reduction of hERG activity. Part 2

The structure-activity relationship (SAR) exploration using 2-(2-furanyl)-7-phenyl[1,2,4]triazolo-[1,5-c]pyrimidin-5-amine (1) as a template led to the identification of a novel class of potent and selective adenosine A2A receptor (AR) antagonists. However, these compounds were found to be associated with significant hERG activity. This report discusses the strategy and outcome of an expanded SAR focused on addressing the hERG liability. As a result, compounds 21 and 24 possess excellent in vitro profiles, highly promising in vivo profiles, and acceptable levels of hERG channel inhibition.     

Hypoxia inhibits maturation and trafficking of hERG K channel protein: Role of Hsp90 and ROS

We previously reported that reactive oxygen species (ROS) generated during hypoxia decrease hERG current density and protein expression in HEK cells stably expressing hERG protein. In the present study, we investigated the molecular mechanisms involved in hypoxia-induced downregulation of hERG protein. Culturing cells at low temperatures and addition of chemical chaperones during hypoxia restored hERG expression and currents to normoxic levels while antiarrhythmic drugs, which selectively block hERG channels, had no effect on hERG protein levels. Pulse chase studies showed that hypoxia blocks maturation of the core glycosylated form in the endoplasmic reticulum (ER) to the fully glycosylated form on the cell surface. Co-immunoprecipitation experiments revealed that hypoxia inhibited interaction of hERG with Hsp90 chaperone required for maturation, which was restored in the presence of ROS scavengers. These results demonstrate that ROS generated during hypoxia prevents maturation of the hERG protein by inhibiting Hsp90 interaction resulting in decreased protein expression and currents.     

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.     

Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). A reduction in the hERG current causes long QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. We reported previously that hERG channels in the plasma membrane undergo vigorous internalization under low K⁺ conditions. In the present study, we addressed whether hERG internalization occurs under normal K⁺ conditions and whether/how internalized channels are recycled back to the plasma membrane. Using patch clamp, Western blot, and confocal imaging analyses, we demonstrated that internalized hERG channels can effectively recycle back to the plasma membrane. Low K⁺-enhanced hERG internalization is accompanied by an increased rate of hERG recovery in the plasma membrane upon reculture following proteinase K-mediated clearance of cell-surface proteins. The increased recovery rate is not due to enhanced protein synthesis, as hERG mRNA expression was not altered by low K⁺ exposure, and the increased recovery was observed in the presence of the protein biosynthesis inhibitor cycloheximide. GTPase Rab11, but not Rab4, is involved in the recycling of hERG channels. Interfering with Rab11 function not only delayed hERG recovery in cells after exposure to low K⁺ medium but also decreased hERG expression and function in cells under normal culture conditions. We concluded that the recycling pathway plays an important role in the homeostasis of plasma membrane-bound hERG channels.     

Bicyclo[3.1.0]hexyl urea melanin concentrating hormone (MCH) receptor-1 antagonists: impacting hERG liability via aryl modifications

Herein, we report the discovery of an effective strategy to modulate liabilities related to affinity of previously disclosed bicyclohexane MCHR-1 antagonists for the hERG channel. This paper describes one of several strategies incorporated to limit hERG binding via modifications of a terminal aryl group in an otherwise promising bicyclohexyl urea series.     

The role of monoubiquitination in endocytic degradation of human ether-a-go-go-related gene (hERG) channels under low K+ conditions

A reduction in extracellular K(+) concentration ([K(+)](o)) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (I(Kr)) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K(+) conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K(+)-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K(+) conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K(+) conditions.     

Structure Driven Design of Novel Human Ether-A-Go-Go-Related-Gene Channel (hERG1) Activators

One of the main culprits in modern drug discovery is apparent cardiotoxicity of many lead-candidates via inadvertent pharmacologic blockade of K⁺, Ca²⁺ and Na⁺ currents. Many drugs inadvertently block hERG1 leading to an acquired form of the Long QT syndrome and potentially lethal polymorphic ventricular tachycardia. An emerging strategy is to rely on interventions with a drug that may proactively activate hERG1 channels reducing cardiovascular risks. Small molecules-activators have a great potential for co-therapies where the risk of hERG-related QT prolongation is significant and rehabilitation of the drug is impractical. Although a number of hERG1 activators have been identified in the last decade, their binding sites, functional moieties responsible for channel activation and thus mechanism of action, have yet to be established. Here, we present a proof-of-principle study that combines de-novo drug design, molecular modeling, chemical synthesis with whole cell electrophysiology and Action Potential (AP) recordings in fetal mouse ventricular myocytes to establish basic chemical principles required for efficient activator of hERG1 channel. In order to minimize the likelihood that these molecules would also block the hERG1 channel they were computationally engineered to minimize interactions with known intra-cavitary drug binding sites. The combination of experimental and theoretical studies led to identification of functional elements (functional groups, flexibility) underlying efficiency of hERG1 activators targeting binding pocket located in the S4–S5 linker, as well as identified potential side-effects in this promising line of drugs, which was associated with multi-channel targeting of the developed drugs.     

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.     

Isoform-Specific Dominant-Negative Effects Associated with hERG1 G628S Mutation in Long QT Syndrome

Background

Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K⁺ channel with properties similar to the rapidly activating delayed rectifying K⁺ current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a_USO isoforms.

Methods

A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation.

Conclusions

The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a_USO isoform. hERG1a_USO-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.     

Enhanced inhibitory effect of acidosis on hERG potassium channels that incorporate the hERG1b isoform

Extracellular acidosis occurs in the heart during myocardial ischemia and can lead to dangerous arrhythmias. Potassium channels encoded by hERG (human ether-à-go-go-related gene) mediate the cardiac rapid delayed rectifier K⁺ current (I_Kr), and impaired hERG function can exacerbate arrhythmia risk. Nearly all electrophysiological investigations of hERG have centred on the hERG1a isoform, although native I_Kr channels may be comprised of hERG1a and hERG1b, which has a unique shorter N-terminus. This study has characterised for the first time the effects of extracellular acidosis (an extracellular pH decrease from 7.4 to 6.3) on hERG channels incorporating the hERG1b isoform. Acidosis inhibited hERG1b current amplitude to a significantly greater extent than that of hERG1a, with intermediate effects on coexpressed hERG1a/1b. I_hERG tail deactivation was accelerated by acidosis for both isoforms. hERG1a/1b activation was positively voltage-shifted by acidosis, and the fully-activated current–voltage relation was reduced in amplitude and right-shifted (by ∼10 mV). Peak I_hERG1a/1b during both ventricular and atrial action potentials was both suppressed and positively voltage-shifted by acidosis. Differential expression of hERG isoforms may contribute to regional differences in I_Kr in the heart. Therefore inhibitory effects of acidosis on I_Kr could also differ regionally, depending on the relative expression levels of hERG1a and 1b, thereby increasing dispersion of repolarization and arrhythmia risk.     

Interaction between the cardiac rapidly (IKr) and slowly (IKs) activating delayed rectifier potassium channels revealed by low K+-induced hERG endocytic degradation

Cardiac repolarization is controlled by the rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier potassium channels. The human ether-a-go-go-related gene (hERG) encodes I(Kr), whereas KCNQ1 and KCNE1 together encode I(Ks). Decreases in I(Kr) or I(Ks) cause long QT syndrome (LQTS), a cardiac disorder with a high risk of sudden death. A reduction in extracellular K(+) concentration ([K(+)](o)) induces LQTS and selectively causes endocytic degradation of mature hERG channels from the plasma membrane. In the present study, we investigated whether I(Ks) compensates for the reduced I(Kr) under low K(+) conditions. Our data show that when hERG and KCNQ1 were expressed separately in human embryonic kidney (HEK) cells, exposure to 0 mM K(+) for 6 h completely eliminated the mature hERG channel expression but had no effect on KCNQ1. When hERG and KCNQ1 were co-expressed, KCNQ1 significantly delayed 0 mM K(+)-induced hERG reduction. Also, hERG degradation led to a significant reduction in KCNQ1 in 0 mM K(+) conditions. An interaction between hERG and KCNQ1 was identified in hERG+KCNQ1-expressing HEK cells. Furthermore, KCNQ1 preferentially co-immunoprecipitated with mature hERG channels that are localized in the plasma membrane. Biophysical and pharmacological analyses indicate that although hERG and KCNQ1 closely interact with each other, they form distinct hERG and KCNQ1 channels. These data extend our understanding of delayed rectifier potassium channel trafficking and regulation, as well as the pathology of LQTS.     

Comparative effects of liensinine and neferine on the human ether-a-go-go-related gene potassium channel and pharmacological activity analysis

Liensinine and neferine, a kind of isoquinoline alkaloid, can antagonize the ventricular arrhythmias. The human ether-a-go-go-related gene (hERG) is involved in repolarization of cardiac action potential. We investigated the effects of liensinine and neferine on the biophysical properties of hERG channel and the underlying structure-activity relationships. The effects of liensinine and neferine were examined on the hERG channels in the stable transfected HEK293 cells using a whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. The pharmacokinetics and tissue distribution determination of liensinine and neferine in rats were determined by a validated RP-HPLC method. Liensinine and neferine induced decrease of current amplitude in dose-dependent. Liensinine reduced hERG tail current from 70.3±6.3 pA/pF in control group to 56.7±2.8 pA/pF in the 1 μM group, 53.0±2.3 pA/pF (3 μM) and 17.8±0.7 pA/pF (30 μM); the corresponding current densities of neferine-treated cells were 41.9±3.1 pA/pF, 32.3±3.1 pA/pF and 16.2±0.6 pA/pF, respectively. Neferine had binding affinity for the open and inactivated state of hERG channel, liensinine only bound to the open state. The inhibitory effects of liensinine and neferine on hERG current were attenuated in the F656V or Y652A mutant channels. Neferine distributed more quickly than liensinine in rats, which was found to be in higher concentration than liensinine. Both liensinine and neferine had no effect on the generation and expression of hERG channels. In conclusion, neferine is a more potent blocker of hERG channels than liensinine at low concentration (<10 μM), which may be due to higher hydrophobic nature of neferine compared with liensinine. Neferine may be safety even for long-term treatment as an antiarrhythmic drug.     

A place for high-throughput electrophysiology in cardiac safety: screening hERG cell lines and novel compounds with the ion works HTTM system

Several commercially available pharmaceutical compounds have been shown to block the IKr current of the cardiac action potential. This effect can cause a prolongation of the electrocardiogram QT interval and a delay in ventricular repolarization. The Food and Drug Administration recommends that all new potential drug candidates be assessed for IKr block to avoid a potentially lethal cardiac arrhythmia known as torsades de pointes. Direct compound interaction with the human ether-a-go-go- related gene (hERG) product, a delayed rectifier potassium channel, has been identified as a molecular mechanism of IKr block. One strategy to identify compounds with hERG liability is to monitor hERG current inhibition using electrophysiology techniques. The authors describe the Ion Works HT instrument as a tool for screening cell lines expressing hERG channels. Based on current amplitude and stability criteria, a cell line was selected and used to perform a 300-compound screen. The screen was able to identify compounds with hERG activity within projects that spanned different therapeutic areas. The cell line selection and optimization, as well as the screening abilities of the Ion Works HT system, provide a powerful means of assessing hERGactive compounds early in the drug discovery pipeline.     

The hERG K(+) channel S4 domain L532P mutation: characterization at 37°C

hERG (human Ether-à-go-go Related Gene) is responsible for ion channels mediating rapid delayed rectifier potassium current, I(Kr), which is key to cardiac action potential repolarization. Gain-of-function hERG mutations give rise to the SQT1 variant of the Short QT Syndrome (SQTS). Reggae mutant zebrafish, with a S4 zERG mutation (Leucine499Proline; L499P), display arrhythmic features analogous to those seen in the SQTS. The affected S4 domain ERG residue is highly conserved. This study was executed to determine how the homologous hERG mutation (L532P) influences channel function at 37°C. Whole-cell measurements of current (I(hERG)) were made from HEK 293 cells expressing WT or L532P hERG. The half maximal activation voltage (V(0.5)) of L532P I(hERG) was positively shifted by ~+36mV compared to WT I(hERG); however at negative voltages a pronounced L532P I(hERG) was observed. Both activation and deactivation time-courses were accelerated for L532P I(hERG). The inactivation V(0.5) for L532P I(hERG) was shifted by ~+32mV. Under action potential (AP) voltage-clamp, L532P I(hERG) exhibited a dome-shaped current peaking at ~+16mV, compared to ~-31mV for WT-I(hERG). The L532P mutation produced an ~5-fold increase in the IC(50) for dronedarone inhibition of I(hERG). Homology modeling indicated that the L532 residue within the S4 helix lies closely apposed to the S5 region of an adjacent hERG subunit. Alterations to the S4 domain structure and, potentially, to interactions between adjacent hERG subunits are likely to account for the functional effects of this mutation.     

hERG1a N-terminal eag domain-containing polypeptides regulate homomeric hERG1b and heteromeric hERG1a/hERG1b channels: a possible mechanism for long QT syndrome

Human ether-á-go-go-related gene (hERG) potassium channels are critical for cardiac action potential repolarization. Cardiac hERG channels comprise two primary isoforms: hERG1a, which has a regulatory N-terminal Per-Arnt-Sim (PAS) domain, and hERG1b, which does not. Isolated, PAS-containing hERG1a N-terminal regions (NTRs) directly regulate NTR-deleted hERG1a channels; however, it is unclear whether hERG1b isoforms contain sufficient machinery to support regulation by hERG1a NTRs. To test this, we constructed a series of PAS domain-containing hERG1a NTRs (encoding amino acids 1-181, 1-228, 1-319, and 1-365). The NTRs were also predicted to form from truncation mutations that were linked to type 2 long QT syndrome (LQTS), a cardiac arrhythmia disorder associated with mutations in the hERG gene. All of the hERG1a NTRs markedly regulated heteromeric hERG1a/hERG1b channels and homomeric hERG1b channels by decreasing the magnitude of the current-voltage relationship and slowing the kinetics of channel closing (deactivation). In contrast, NTRs did not measurably regulate hERG1a channels. A short NTR (encoding amino acids 1-135) composed primarily of the PAS domain was sufficient to regulate hERG1b. These results suggest that isolated hERG1a NTRs directly interact with hERG1b subunits. Our results demonstrate that deactivation is faster in hERG1a/hERG1b channels compared to hERG1a channels because of fewer PAS domains, not because of an inhibitory effect of the unique hERG1b NTR. A decrease in outward current density of hERG1a/hERG1b channels by hERG1a NTRs may be a mechanism for LQTS.     

Proton block of the pore underlies the inhibition of hERG cardiac K+ channels during acidosis

Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.     

Isosteric replacements for benzothiazoles and optimisation to potent Cathepsin K inhibitors free from hERG channel inhibition

The discovery of nitrile compound 4, a potent inhibitor of Cathepsin K (Cat K) with good bioavailability in dog is described. The compound was used to demonstrate target engagement and inhibition of Cat K in an in vivo dog PD model. The margin to hERG ion channel inhibition was deemed too low for a clinical candidate and an optimisation program to find isosteres or substitutions on benzothiazole group led to the discovery of 20, 24 and 27; all three free from hERG inhibition.     

Cell Surface Expression of Human Ether-a-go-go-related Gene (hERG) Channels Is Regulated by Caveolin-3 Protein via the Ubiquitin Ligase Nedd4-2

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (I_Kr) which plays an important role in cardiac repolarization. A reduction or increase in hERG current can cause long or short QT syndrome, respectively, leading to fatal cardiac arrhythmias. The channel density in the plasma membrane is a key determinant of the whole cell current amplitude. To gain insight into the molecular mechanisms for the regulation of hERG density at the plasma membrane, we used whole cell voltage clamp, Western blotting, and immunocytochemical methods to investigate the effects of an integral membrane protein, caveolin-3 (Cav3) on hERG expression levels. Our data demonstrate that Cav3, hERG, and ubiquitin-ligase Nedd4-2 interact with each other and form a complex. Expression of Cav3 thus enhances the hERG-Nedd4-2 interaction, leading to an increased ubiquitination and degradation of mature, plasma-membrane localized hERG channels. Disrupting Nedd4-2 interaction with hERG by mutations eliminates the effects of Cav3 on hERG channels. Knockdown of endogenous Cav3 or Nedd4-2 in cultured neonatal rat ventricular myocytes using siRNA led to an increase in native I_Kr. Our data demonstrate that hERG expression in the plasma membrane is regulated by Cav3 via Nedd4-2. These findings extend our understanding of the regulation of hERG channels and cardiac electrophysiology.     

hERG1 Potassium Channel Expression in Colorectal Adenomas: Comparison with Other Preneoplastic Lesions of the Gastrointestinal Tract

Preneoplastic lesions represent a useful target for early diagnosis and follow-up of gastrointestinal malignancies. hERG1 channel expression was tested by immunohistochemistry (IHC) in a cohort of colorectal adenoma samples belonging to Italian subjects. Overall, hERG1 was expressed in 56.5% of cases with both high staining intensity and a high percentage of positive cells. Moreover, hERG1 was expressed in a higher percentage of dysplastic adenomas with respect to hyperplastic lesions, and the proportion of positive samples further increased in patients with high-grade dysplasia. Comparing hERG1 expression in other preneoplastic lesions of the GI tract (gastric dysplasia and Barrett’s esophagus), it emerged that in all the conditions, hERG1 was expressed with a diffused pattern, throughout the cell, with variable staining intensity within the samples. The highest expression was detected in gastric dysplasia samples and the lowest in Barrett’s esophagus at similar levels observed in colorectal adenomas. Our results show that hERG1 is aberrantly expressed in human preneoplastic lesions of the gastrointestinal tract and has a different pattern of expression and role in the different sites. Overall, the detection of hERG1 expression in preneoplastic lesions could represent a novel diagnostic or prognostic marker of progression in the gastrointestinal tract.