Biology subjects

Alameh, M.

Publications and source records attributed to Alameh, M..

4 recordsLinked to original sources

bioRxiv2026

Expression-System Biases Compromise the Functional Assessment of KCNH2 Variants and Clinical Risk Assessment

Background: Functional characterization of KCNH2 variants is essential for assessing pathogenicity in long QT syndrome type 2. Because hERG channels assemble as tetramers, the relative expression of wild-type (WT) and variant subunits critically influences channel function and pathogenicity assessment. However, commonly used experimental systems may not reproduce the balanced allelic expression observed in heterozygous patients. Methods: Allele-specific KCNH2 expression was analyzed in two independent RNA-sequencing datasets from human left ventricular tissue. Using the dominant-negative T74R hERG variant as a model, we calibrated the relationship between WT-to-variant expression ratios and hERG current amplitude. Using automated patch-clamp electrophysiology, we compared 3 dual-expression strategies: internal ribosome entry site (IRES), P2A-linked tandem constructs, and dual cytomegalovirus promoter (dCMV) constructs. Co-electroporation of WT and variant plasmids in different ratios was the reference. Results: Allele-specific expression analysis supported balanced KCNH2 expression in human left ventricular tissue. At a 1:1 WT-to-T74R expression ratio, hERG current amplitude was reduced to 31.0% of WT, consistent with a dominant-negative mechanism in which incorporation of 2 or more T74R subunits renders the channel nonfunctional. All 3 dual-expression strategies produced substantial expression imbalances, with the first cassette consistently favored. IRES constructs showed the greatest asymmetry. P2A constructs partially improved expression balance but introduced an additional N-terminal proline in the second cassette altering T74R-associated trafficking and channel biophysics. The dCMV system also failed to achieve balanced expression, including after stable integration. Co-electroporation of separate WT and variant plasmids provided the most reliable means of controlling their relative expression. Conclusions: Commonly used dual-expression constructs do not reliably reproduce balanced WT and variant hERG expression, potentially distorting assessments of dominant-negative effects and variant pathogenicity. Co-electroporation of separate plasmids provides a robust approach for high-throughput functional phenotyping under heterozygous conditions, with implications for the interpretation of KCNH2 variants and potentially other genes encoding multimeric ion channels.

biophysics↗

bioRxiv2026

MeTAL enables multiparametric risk prediction for human KCNH2 variants

BackgroundClinical interpretation of missense variants in the hERG potassium channel encoded by the KCNH2 gene remains a major challenge in inherited arrhythmia syndromes. Functional studies often rely on a minimal set of channel properties, mainly current amplitude measurements, which do not capture the multidimensional nature of channel gating and its impact on ventricular repolarization. We developed a multiscale computational framework to quantitatively link multiparametric channel dysfunction to ECG phenotypes. MethodsWe generated multiparametric electrophysiological profiles for KCNH2 variants using high-throughput patch-clamp, quantifying nine biophysical properties including conductance, voltage dependence, and gating kinetics. These parameters were incorporated into a modified formulation of IKr embedded in a human ventricular electrophysiology model. The resulting framework, termed MeTAL (Multiscale-enriched Transformation and Analysis for Long-QT), produces physiologically calibrated pseudo-ECGs enabling quantitative evaluation of QT dynamics. We systematically analyzed the contribution of individual and combined gating parameters and applied the model to 41 KCNH2 variants across ACMG classes, comparing simulated QTc values with clinical data. ResultsMultiparametric profiling revealed complex functional signatures in most variants, with concurrent gain- and loss-of-function effects affecting distinct gating processes. Simulations demonstrated that ventricular repolarization, though strongly determined by current amplitude, is substantially influenced by inactivation-related parameters, particularly the slope and voltage dependence of inactivation. Interaction analyses showed nonlinear relationships between gating parameters, explaining why variants with similar current density can produce divergent QT phenotypes. In heterozygous simulations, MeTAL reproduced clinically observed QTc distributions across variant classes and accurately predicted the repolarization regime (normal, long-QT, or short-QT) in most cases. ConclusionsMultiparametric integration of ion-channel function within a multiscale electrophysiological model enables mechanistic prediction of QT behavior beyond conductance-based metrics. This approach provides a scalable framework for interpretation of KCNH2 variants and solves the issue of risk stratification in inherited arrhythmia syndromes while offering new opportunities for variant-specific and pharmacological modeling of repolarization.

biophysics↗

bioRxiv2023

Predicting hERG repolarization power at 37°C from recordings at room temperature

Mutations in the KCNH2 gene cause long or short QT syndromes (LQTS or SQTS) predisposing to life-threatening arrhythmias. KCNH2 encodes for the voltage-gated K+ channel hERG involved in the late repolarization phase of the cardiac action potential (AP). For the last decades, sequencing KCNH2 has provided a plethora of variants associated or not with clear pathological cardiac phenotypes. Identifying pathogenic or likely pathogenic variants from the benign ones would provide useful information to clarify the genetic background of LQTS patients and relatives, and to stratify the risk of adverse events. In face of a wide spectrum of hERG biophysical defects, we looked for a way to summarize the net loss or gain of function in a unique index. In a previous work, we defined as the repolarization power the time integral of the K+ currents developed during an AP clamp. Here, with the aim of accelerating the functional characterization of hERG variants using automated patch-clamp, we adapted the AP-clamp protocol to establish, at room temperature, at which the recording success rate is high, a repolarization power index, as reliable and informative as the one measured at physiological temperature. We also illustrate that the repolarization power determined at room temperature is predictive of the repolarization power at physiological temperature for 2 pathogenic hERG variants with different biophysical dysfunctions.

physiology↗

bioRxiv2022

SARS-CoV2 E and 3a proteins are inducers of pannexin currents

Controversial reports have suggested that SARS-CoV E and 3a proteins may be viroporins that conduct currents through the plasma membrane of the infected cells. If true, these proteins would represent accessible targets for the development of new antiviral drugs by using high-throughput patch-clamp techniques. Here we aimed at better characterizing the cell responses induced by E or 3a protein with a particular focus on the ion conductances measured at the cell surface. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly-acquired round shape, tending to detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this hypothesis by using flow cytometry, in agreement with earlier reports on other cell types. In adhering cells expressing E or 3a protein, whole-cell currents were in fact not different from the control condition indicating that E and 3a proteins are not plasma membrane viroporins. In contrast, recording currents on detached cells uncovered outwardly-rectifying currents, much larger than those observed in control. The current characteristics are reminiscent of what was previously observed in cells expressing SARS-CoV-1 E or 3a proteins. Herein, we illustrate for the first time that carbenoxolone blocks these outward currents suggesting that they are conducted by pannexin channels, mostly likely activated by cell morphology change and/or cell death. Alongside we also demonstrate that truncation of the C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent pannexin currents suggesting distinct pathways for cell death and pannexin currents induced by E and 3a proteins. We conclude that SARS-CoV-2 E and 3a proteins are not acting as viroporins expressed at the plasma membrane. Author SummaryA viroporin (or viral porin) is a class of proteins that is encoded by a virus genome. It is named porin because its biological role is to conduct ions through a pore that it created in a lipid membrane such as the one surrounding a human cell. if such viroporin is present at the external membrane of a human cell infected by a virus, it can be an easy target of an antiviral agent which thus does not have to enter the cell to be active. One example of viroporin is the flu M2 protein that is the target of amantadine, an antiviral agent used against flu. In previous studies, two proteins of SARS-CoV viruses, named E protein and 3a protein, have been suggested to be viroporins at the surface of infected human cells, potentially opening a new research avenue against SARS. Here we demonstrate that both proteins are not viroporins at the external membrane but they rather trigger changes in the cell shape and promote cell death. They only indirectly induce the activity of a porin that is encoded by the cell genome, named pannexin.

cell biology↗