Mito Thermo Yellow (MTY) is a mitochondrially targeted fluorophore that shows marked fluorescence quenching with increasing temperature, allowing for interrogating temperature dynamics in the mitochondria of live cells. Here we re-evaluate published MTY fluorescence responses used to argue in favor of the 'hot mitochondria' concept; the assertion that mitochondria operate while maintaining substantial (> 10°C) apparent temperature gradients (ΔTapp) between themselves and their cellular environment. We find that MTY fluorescence kinetics are incompatible with the expected dynamics of mitochondrial heat production and diffusion. We further explore the published effects of mitochondrial inhibitors on MTY, and related evidence for ΔTapp of > 10°C, again concluding results are inconsistent with the expected heat production dynamics. Thus, assertions of ΔTapp > 10°C between mitochondria and their cellular environment based on MTY fluorescence intensity changes are unlikely to be reporting a signal that is uniquely intramitochondrial temperature. In addition to these analyses, we further argue that the inference mitochondria can operate at an internal temperature of > 48°C, as reported using MTY, is improbable as these internal temperatures would cause protein denaturation and aggregation and induction of the heat shock (HSR), unfolded protein (UPR), and integrated (ISR) stress responses. Taken as a whole, we conclude MTY and similar tools must be re-evaluated in regard to if they are providing solely information on local temperature and thus are so far inadequate, unto themselves, to demonstrate the existence of hot mitochondria.

Loss-of-function mutations in KCNJ10, encoding Kir4.1, cause EAST/SeSAME syndrome, with renal salt-wasting tubulopathy and hypokalemia. We hypothesized that Kir4.1 deletion specifically in the distal convoluted tubule (DCT) stimulates ENaC activity via the mammalian target of rapamycin (mTOR)-dependent mechanisms, contributing to hypokalemia. Metabolic cages, electrophysiology, immunoblotting, immunostaining, and invivo diuretic response experiments were used to examine biochemical parameters, Kir4.1/Kir5.1 activity, NCC and ENaC function in the DCT-specific Kir4.1 knockout (DCT-Kir4.1 KO) mice under normal or K+ restriction conditions. DCT-Kir4.1 KO mice exhibited impaired basolateral K+ channel and NCC activity, enhanced ENaC activity, and mild hypokalemia. Amiloride treatment induced similar natriuresis and kaliuresis in DCT-Kir4.1 KO and kidney-specific Kir4.1 KO mice, but had minimal effects in collecting system Kir4.1 KO mice, suggesting high ENaC activity following Kir4.1 deletion in the DCT. Notably, severe hypokalemia, along with upregulated ENaC expression and activity, was observed in DCT-Kir4.1 KO mice under dietary K+ restriction. Patch-clamp experiments further revealed elevated ENaC currents in the DCT2 of KO mice on a low-K+ diet, independent of aldosterone levels. Inhibition of mTOR with AZD8055 reduced SGK1/Nedd4-2 phosphorylation, cleaved α-ENaC expression, and DCT2 ENaC currents, suggesting a role for mTOR in ENaC hyperactivity in K+-restricted DCT-Kir4.1 KO mice. This notion was also supported by the upregulated Rictor expression observed in the isolated DCT of these KO mice. We conclude that Kir4.1 deletion drives ENaC hyperactivity in the DCT via the mTORC2-dependent SGK1/Nedd4-2 signaling pathway, promoting low potassium diet-induced hypokalemia.

The tandem pore domain halothane-inhibited potassium (THIK-1) channel is a member of the two-pore domain potassium (K2P) channel family and plays a critical role in maintaining the resting membrane potential. THIK-1 has emerged as a key regulator of microglial physiology and neuroimmune signaling. With the rapid accumulation of structural, electrophysiological, and functional evidence, there is an increasing need for an integrated understanding of THIK-1 in the context of microglial biology and disease. This review provides a comprehensive synthesis of the structural, regulatory, and functional properties of THIK-1, with a particular focus on its roles in microglial physiology, neuroimmune signaling, and central nervous system (CNS) pathologies. We conducted a comprehensive review of recent literature, including electrophysiological, molecular, and structural studies, with particular emphasis on cryo-electron microscopy findings, pharmacological modulation, and disease-associated functional analyses. THIK-1 is selectively enriched in microglia and contributes to essential cellular processes, including surveillance motility, synaptic pruning, and inflammasome activation. Its high constitutive activity makes it a dominant determinant of the microglial membrane potential. Structural studies have identified key features, including a lipid-interacting pocket and a cytoplasmic gate, which underlie lipid- and anesthetic-mediated regulation. Functionally, THIK-1-mediated K⁺ efflux is required for NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation and pyroptosis. Accumulating evidence links THIK-1 to major CNS disorders, including neuroinflammation, neurodegeneration (e.g., Alzheimer's and Parkinson's diseases), and psychiatric disorders. The convergence of structural, electrophysiological, and immunological findings positions THIK-1 as a central regulator of neuroimmune signaling. Integration of these findings provides new insights into how ion channel activity shapes microglial function and disease processes. THIK-1 represents a critical nexus between ion channel biophysics and neuroimmune dysfunction. A comprehensive understanding of its regulation and function supports its potential as a microglia-specific therapeutic target in neuroinflammatory and neurodegenerative disorders.

ABSTRACTAimRenal sodium reabsorption occurs through both transcellular and paracellular pathways. Tight junction proteins play a key role in mediating paracellular transport. The collecting duct is critical for the fine tuning of sodium balance and is highly responsive to changes in dietary salt intake. This study aimed to determine whether a low‐sodium diet modulates paracellular sodium permeability by regulating the expression or localization of claudin‐3, a major tight junction protein in the collecting duct.MethodsWild‐type and claudin‐3 knockout male mice were fed low (0.01%) or normal (0.18%) sodium diets for 7 days, with or without treatment with spironolactone, a mineralocorticoid receptor antagonist. The expression of tight junction proteins was analyzed by immunoblotting and immunofluorescence. Functional effects of claudin‐3 on ion permeability were evaluated in cultured mouse collecting duct principal cells using chamber recordings after claudin‐3 overexpression or gene silencing.ResultsLow‐sodium diet increases claudin‐3 expression in mouse kidneys. In cultured cells, aldosterone enhanced claudin‐3 abundance and its plasma membrane localization. Claudin‐3 overexpression reduced, while its silencing increased paracellular permeability to sodium and chloride. Claudin‐3 knockout mice on a low‐sodium diet compensated by upregulating epithelial sodium channel subunits, claudin‐4, claudin‐8, and claudin‐10. This adaptive response persisted under mineralocorticoid receptor blockade.ConclusionsOur findings demonstrate that aldosterone strengthens the paracellular sodium barrier in the collecting duct by inducing claudin‐3. In the absence of claudin‐3, compensatory regulation of other claudins and sodium transporters preserves sodium homeostasis under low‐salt conditions, thus revealing adaptive mechanisms in renal sodium handling.