Tsai, P. | bioRxiv Science

bioRxiv2026

A Brugada-related KCNT1 mutation unveils its conductance-independent activation of store-operated Ca 2+ entry

BackgroundPotassium sodium-activated channel subfamily T member 1 (KCNT1) is a Na+-activated K+ channel, associated with epilepsy and cardiac diseases. Besides K+ conductance, KCNT1 is also involved in Ca2+ handling. KCNT1:R1106Q was identified in a Brugada Syndrome (BrS) patient; however, its pathological characteristics regarding either K+ or Ca2+ homeostasis remain to be fully elucidated. MethodsFura-2-loaded HEK293T cells were used for intracellular Ca2+ investigations, and whole-cell patch-clamp technique were used as complementary tests. Stromal interaction molecule 1 (STIM1) aggregation and endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HeLa cells were visualized using spinning disc confocal microscope. Multielectrode array (MEA) assays were utilized to assess the field potential duration (FPD) and spontaneous beating rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ResultsKCNT1 overexpression increased SOCE, which was further upregulated by the KCNT1R1106Q mutant. Furthermore, KCNT1 upregulated ER Ca2+ release and was found to localize at ER-PM junctions. The ER Ca2+ dynamics in KCNT1R1106-overexpressing cells was disrupted, while its SOCE remained intact. The KCNT1 cytoplasmic tail alone sufficiently modulated Ca2+ homeostasis. Given that KCNT1 possesses prominent charge-concentrated segments within its cytoplasmic domain, we substituted charged amino acids in either the 740-DDE-742 or 1114-RRLSR-1118 motifs with alanine; these substitutions abolished the KCNT1-mediated increases in ER Ca2+ release and ER-PM contact formation. MEA recordings of hiPSC-CMs revealed that the overexpression of KCNT1 shortened the FPD and accelerated the cardiomyocyte beating rate compared to baseline. However, the magnitude of beating rate acceleration induced by KCNT1R1106Q was significantly attenuated compared to that of KCNT1WT. ConclusionsOur findings indicate that, independent of KCNT1-mediated ion conductance, the charged motifs located at both ends of the KCNT1 cytoplasmic tail serve as structural anchors to facilitate ER-PM contact formation. This non-conducting role endows KCNT1 with a K+ current-independent mechanism to modulate intracellular Ca2+ homeostasis and cardiomyocyte physiology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/731813v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@104e234org.highwire.dtl.DTLVardef@19eceb3org.highwire.dtl.DTLVardef@1148512org.highwire.dtl.DTLVardef@2c8118_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and SignificanceO_ST_ABSWhat is known?C_ST_ABSO_LIKCNT1 is a Na+-activated K+ channel, and mutations within this gene are often associated with epilepsy and epilepsy-related cardiac diseases. C_LIO_LIBesides functioning as a rectifier, KCNT1 is implicated in cellular Ca2+ handling, such as regulating Ca2+ oscillations in rat cortical neurons. C_LIO_LIKCNT1:R1106Q missense mutation has been identified in a patient with Brugada Syndrome (BrS); however, its pathophysiological characteristics and underlying mechanisms remain uncharacterized. C_LI What New Information Does This Article Contribute?O_LIKCNT1 overexpression upregulates both ER Ca2+ release and store-operated Ca2+ entry (SOCE). KCNT1R1106Q further increased SOCE intensity, however, its increase in ER Ca2+ release and [Ca2+]ER were attenuated. C_LIO_LIThe cytoplasmic tail of KCNT1 alone is suNicient to modulate Ca2+ closely mimicking the eNects of full-length KCNT1. C_LIO_LIThe charged clusters distributed along the KCNT1 cytoplasmic tail facilitate ER-PM contact. Alanine substitutions abolish the enhancements in ER Ca2+ release and ER-PM contact formation, while SOCE modulation remains intact. C_LIO_LIThe KCNT1 cytoplasmic tail alone shortens the field potential duration (FPD) and accelerates the spontaneous beating rate in hiPSC-CMs. However, the magnitude of beating rate acceleration induced by KCNT1R1106Q is significantly attenuated compared to KCNT1WT. C_LI This study uncovers a novel, conductance-independent role for the KCNT1 channel in structural organization, demonstrating that the charged motifs on its cytoplasmic tail serve as physical anchors facilitating ER-PM junctions. This structural mechanism regulates intracellular Ca2+ homeostasis and subsequently alters cardiomyocyte electrophysiology. By decoupling KCNT1s structural impact from its ion-conducting function, these findings provide new mechanistic insights into how specific KCNT1 variants, such as the BrS-associated KCNT1:R1106Q mutation, may contribute to arrhythmogenesis through imbalanced Ca2+ handling rather than classical K+ current alterations.

Molecular Biology↗

bioRxiv2023

Myofascial Trigger Point (MTrP) Size and Elasticity Properties Can Be Used to Differentiate Active and Latent MTrPs in Lower Back Skeletal Muscle

Myofascial Trigger Points (MTrPs) are localized contraction knots that develop after muscle overuse or an acute trauma. Significant work has been done to understand, diagnose, and treat MTrPs in order to improve patients suffering from their effects. However, effective non-invasive diagnostic tools are still a missing gap in both understanding and treating MTrPs. Effective treatments for patients suffering from MTrP mediated pain require a means to measure MTrP properties quantitatively and diagnostically both prior to and during intervention. Further, quantitative measurements of MTrPs are often limited by the availability of equipment and training. Here we develop ultrasound (US) based diagnostic metrics that clinicians can use during patient diagnosis and treatment. We highlight the advantages and limitations of previous US-based approaches that utilize elasticity theory. We show how US-based measurements can distinguish Active from Latent MTrPs. We demonstrate that Active MTrPs tend to be smaller, stiffer, and deeper in the muscle tissue. We provide evidence that more than one MTrP within a single US-image field increases the stiffness of neighboring MTrPs. Finally, we highlight a combination of metrics (depth, thickness, and stiffness) that can be used to assess individual MTrPs in combination with standard clinical assessments.

biophysics↗

bioRxiv2017

Recurrent loss of heterozygosity correlates with clinical outcome in pancreatic neuroendocrine cancer

Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Analysis of gene mutation, copy number and RNA expression of 57 sporadic pNETs showed that pNET genomes are dominated by aneuploidy. Remarkably, ~25% of pNETs had genomes characterized by recurrent loss of heterozygosity (LoH) of the same 10 chromosomes, accompanied by bi-allelic MEN1 inactivation, and these cases had generally poor clinical outcome. Another ~25% of all pNETs had chromosome 11 LoH and bi-allelic MEN1 inactivation, lacking the recurrent LoH pattern - these had universally good clinical outcome. Some level of aneuploidy was common, and overall ~80% of pNETs had LoH of [≥]1 chromosome. This aneuploidy led to changes in RNA expression at the level of whole chromosomes and allowed pathogenic germline variants (e.g. ATM) to be expressed unopposed, inactivating downstream tumor suppressor pathways. Some pNETs appear to utilize VHL gene methylation or mutation to activate pseudo-hypoxia. Contrary to expectation neither tumor morphology within well-differentiated pNETs nor single gene mutation had significant associations with clinical outcome, nor did expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. MEN1 was the only statistically significant recurrently mutated driver gene in pNETs. Only one pNET had clearly oncogenic and actionable SNVs (in PTEN and FLCN) confirmed by corroborating RNA expression changes. The two distinct patterns of aneuploidy described here, associated with markedly poor and good clinical outcome respectively, define a novel oncogenic mechanism and the first route to genomic precision oncology for this tumor type.

cancer biology↗