Biology subjects

Desai, R. R.

Publications and source records attributed to Desai, R. R..

5 recordsLinked to original sources

bioRxiv2025

Classification models distinguish functional and trafficking effects of KCNQ1 variants to enhance variant interpretation

Missense mutations compromise protein fitness by altering stability and function, which can lead to various clinical disease states. The potassium ion channel KCNQ1 underlies the majority of congenital long QT syndrome (LQTS) cases, one of the most common genetic arrhythmia syndromes. During genetic testing for LQTS, variants of uncertain significance (VUS) confound diagnosis and clinical management. KCNQ1 protein fitness metrics enable mechanistic classification of variants, directly informing the molecular basis for dysfunction and providing clinical interpretation of variants linked to LQTS and other channelopathies. We developed structure-aware random forest classifier models to predict seven metrics of KCNQ1 fitness, four functional electrophysiology measurements (peak current density, voltage-dependence, gating kinetics), and three trafficking values measured by flow cytometry. Our trained models outperformed AlphaMissense in predicting protein fitness, enhancing interpretation of ClinVar VUS and variants classified as ambiguous by AlphaMissense. We demonstrate the classifiers distinguish benign and pathogenic variants from ClinVar and gnomAD and identify systematic patterns of dysfunction and mistrafficking along the functionally critical S4 helix. Our method advances variant effect prediction with a mechanistic classifier that reliably links missense mutations in KCNQ1 to their specific disease-causing mechanisms. As a resource for precision medicine approaches for LQTS or other KCNQ1 channelopathies, we provide the predictions and scores for all KCNQ1 missense variants across the structured region of the protein.

biophysics↗

bioRxiv2025

Mapping the spatial architecture of glioblastoma from core to edge delineates niche-specific tumor cell states and intercellular interactions

Treatment resistance in glioblastoma (GBM) is largely driven by the extensive multi-level heterogeneity that typifies this disease. Despite significant progress toward elucidating GBMs genomic and transcriptional heterogeneity, a critical knowledge gap remains in defining this heterogeneity at the spatial level. To address this, we employed spatial transcriptomics to map the architecture of the GBM ecosystem. This revealed tumor cell states that are jointly defined by gene expression and spatial localization, and multicellular niches whose composition varies along the tumor core-edge axis. Ligand-receptor interaction analysis uncovered a complex network of intercellular communication, including niche- and region-specific interactions. Finally, we found that CD8 positive GZMK positive T cells colocalize with LYVE1 positive CD163 positive myeloid cells in vascular regions, suggesting a potential mechanism for immune evasion. These findings provide novel insights into the GBM tumor microenvironment, highlighting previously unrecognized patterns of spatial organization and intercellular interactions, and novel therapeutic avenues to disrupt tumor-promoting interactions and overcome immune resistance.

cancer biology↗

bioRxiv2025

Functional profiling of KCNE1 variants informs population carrier frequency of Jervell and Lange-Nielsen syndrome type 2

Congenital long-QT syndrome (LQTS) is most often associated with pathogenic variants in KCNQ1 encoding the pore-forming voltage-gated potassium channel subunit of the slow delayed rectifier current (IKs). Generation of IKs requires assembly of KCNQ1 with an auxiliary subunit encoded by KCNE1, which is also associated with LQTS but causality of autosomal dominant disease is disputed. By contrast, KCNE1 is an accepted cause of recessive type 2 Jervell and Lange-Nielson syndrome (JLN2). The functional consequences of most KCNE1 variants have not been determined and the population prevalence of JLN2 is unknown. Methods: We determined the functional properties of 95 KCNE1 variants co-expressed with KCNQ1 in heterologous cells using high-throughput voltage-clamp recording. Experiments were conducted with each KCNE1 variant expressed in the homozygous state and then a subset was studied in the heterozygous state. The carrier frequency of JLN2 was estimated by considering the population prevalence of dysfunctional variants. Results: There is substantial overlap between disease-associated and population KCNE1 variants. When examined in the homozygous state, 68 KCNE1 variants exhibited significant differences in at least one functional property compared to WT KCNE1, whereas 27 variants did not significantly affect function. Most dysfunctional variants exhibited loss-of-function properties. We observed no evidence of dominant-negative effects. Most variants were scored as variants of uncertain significance (VUS) and inclusion of functional data resulted in revised classifications for only 14 variants. The population carrier frequency of JLN2 was calculated as 1 in 1034. Peak current density and activation voltage-dependence but no other biophysical properties were correlated with findings from a mutational scan of KCNE1. Conclusions: Among 95 disease-associated or population KCNE1 variants, many exhibit abnormal functional properties but there was no evidence of dominant-negative behaviors. Using functional data, we inferred a population carrier frequency for recessive JLN2. This work helps clarify the pathogenicity of KCNE1 variants.

genetics↗

bioRxiv2022

Molecular simulations reveal a mechanism for enhanced allosteric coupling between voltage-sensor and pore domains in KCNQ1 explaining its activation by ML277

The voltage-gated potassium channel KCNQ1 (KV7.1) is important for the repolarizing phase of the cardiac action potential. Activators of KCNQ1 may provide a strategy for the pharmacological treatment of congenital long QT syndrome, a genetic disorder caused by pathogenic variants in KCNQ1 that promote arrhythmia susceptibility and elevate risk for sudden cardiac death. The small-molecule agonist ML277 recovers function of mutant KCNQ1 channels in human induced pluripotent stem cell-derived cardiomyocytes and could represent a starting point for drug development. Here we investigated ML277 mode of action by developing a molecular model of the KCNQ1-ML277 interaction corroborated by experimental and computational analyses. Ligand docking and molecular dynamics simulation demonstrated that ML277 binds to the interface between the voltage sensor and pore domains in KCNQ1. Model predicted binding energies for ML277 and 62 chemical analogs of ML277 correlated with EC50 data available for these compounds. We identified novel ML277-interacting residues on the S5 and S6 segments of KCNQ1 by performing MM/PBSA energy calculations and site-directed mutagenesis of KCNQ1 coupled to electrophysiological characterization of the generated channel mutants. Network analysis of the molecular dynamics simulations further showed that ML277 increases the allosteric coupling efficiency between residues in the voltage sensor domain and residues in the pore domain. Derivatives of ML277 that are not active on KCNQ1 fail to increase allosteric coupling efficiency in the computational simulations. Our results reveal atomic details of the ML277 modulation of KCNQ1 activation. These findings may be useful for the design of allosteric modulators of KCNQ1 and other KCNQ channels that bind at the membrane-accessible protein surface. Statement of SignificanceThe potassium ion channel KCNQ1 contributes to the generation of electrical impulses in the heart. Heritable mutations in KCNQ1 can cause channel loss-of-function and predispose to a life-threatening cardiac arrhythmia. Small molecules that bind KCNQ1 and enhance channel function could establish a novel anti-arrhythmic drug paradigm. We used molecular simulations to investigate how a small agonist of KCNQ1 (ML277) binds to the KCNQ1 channel and increases its function. We identified amino acids that are responsible for ML277 binding and show how ML277 promotes signaling in KCNQ1 and channel opening. This work advances our understanding how KCNQ1 and possibly other potassium channels can be activated with small molecules. These data provide a framework for drug development studies.

biophysics↗

bioRxiv2021

High-throughput Evaluation of Epilepsy-associated KCNQ2 Variants Reveals Functional and Pharmacological Heterogeneity

Hundreds of KCNQ2 variants have been identified by genetic testing of children with early onset epilepsy and/or developmental disability. Voltage-clamp recording from heterologous cells has proved useful for establishing deleterious functional effects of KCNQ2 variants, but procedures adapting these assays for standardized, higher throughput data collection and reporting are lacking. In this study, we employed automated patch clamp recording to assess in parallel the functional and pharmacological properties of 79 missense and 2 in-frame deletion variants of KCNQ2. Among the variants we studied were a training set of 18 pathogenic variants previously studied by voltage-clamp recording, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. Variant KCNQ2 subunits were transiently expressed in a cell line stably expressing KCNQ3 to reconstitute the physiologically relevant channel complex. Variants with severe loss-of-function were also co-expressed 1:1 with WT KCNQ2 in the KCNQ3 cell line to mimic the heterozygous genotype and assess dominant-negative behavior. In total, we analyzed electrophysiological data recorded from 9,480 cells. The functional properties of WT KCNQ2/KCNQ3 channels and pharmacological responses to known blockers and activators determined by automated patch clamp recording were highly concordant with previous findings. Similarly, functional properties of 18 known pathogenic variants largely matched previously published results and the validated automated patch clamp assay. Many of the 39 previously unstudied disease-associated KCNQ2 variants exhibited prominent loss-of-function and dominant-negative effects, providing strong evidence in support of pathogenicity. All variants, exhibit response to retigabine (10 {micro}M), although there were differences in maximal responses. Variants within the ion selectivity filter exhibited the weakest responses whereas retigabine had the strongest effect on gain-of-function variants in the voltage-sensor domain. Our study established a high throughput method to detect deleterious functional consequences of KCNQ2 variants. We demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants but this does not occur with rare population variation in this gene. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine.

neuroscience↗