A Brugada-related KCNT1 mutation unveils its conductance-independent activation of store-operated Ca 2+ entry
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Potassium 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 Ca 2+ handling. KCNT1:R1106Q was identified in a Brugada Syndrome (BrS) patient; however, its pathological characteristics regarding either K + or Ca 2+ homeostasis remain to be fully elucidated.
Methods
Fura-2-loaded HEK293T cells were used for intracellular Ca 2+ 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).
Results
KCNT1 overexpression increased SOCE, which was further upregulated by the KCNT1 R1106Q mutant. Furthermore, KCNT1 upregulated ER Ca 2+ release and was found to localize at ER-PM junctions. The ER Ca 2+ dynamics in KCNT1 R1106 –overexpressing cells was disrupted, while its SOCE remained intact. The KCNT1 cytoplasmic tail alone sufficiently modulated Ca 2+ 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 Ca 2+ 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 KCNT1 R1106Q was significantly attenuated compared to that of KCNT1 WT .
Conclusions
Our 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 Ca 2+ homeostasis and cardiomyocyte physiology.
Novelty and Significance
What is known?
KCNT1 is a Na + -activated K + channel, and mutations within this gene are often associated with epilepsy and epilepsy-related cardiac diseases.
Besides functioning as a rectifier, KCNT1 is implicated in cellular Ca 2+ handling, such as regulating Ca 2+ oscillations in rat cortical neurons.
KCNT1:R1106Q missense mutation has been identified in a patient with Brugada Syndrome (BrS); however, its pathophysiological characteristics and underlying mechanisms remain uncharacterized.
What New Information Does This Article Contribute?
KCNT1 overexpression upregulates both ER Ca 2+ release and store-operated Ca 2+ entry (SOCE). KCNT1 R1106Q further increased SOCE intensity, however, its increase in ER Ca 2+ release and [Ca 2+ ] ER were attenuated.
The cytoplasmic tail of KCNT1 alone is suNicient to modulate Ca 2+ closely mimicking the eNects of full-length KCNT1.
The charged clusters distributed along the KCNT1 cytoplasmic tail facilitate ER-PM contact. Alanine substitutions abolish the enhancements in ER Ca 2+ release and ER-PM contact formation, while SOCE modulation remains intact.
The 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 KCNT1 R1106Q is significantly attenuated compared to KCNT1 WT .
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 KCNT1’s 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.
