Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

Šeflová, Jaroslava; Habibi, Nima R.; Yap, John Q.; Cleary, Sean R.; Fang, Xuan; Kekenes‐Huskey, Peter M.; Espinoza-Fonseca, L. Michel; Bossuyt, Julie; Robia, Seth L.

doi:10.1016/j.jbc.2022.101865

Cited by 14 publications

(9 citation statements)

References 75 publications

(105 reference statements)

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“…The full-length wildtype cDNA for the YFP N-terminally conjugated α1 and the β1 subunits (a gift from Seth L. Robia at Loyola University Chicago) have been previously described ( 42 ). The Y148* point variant was introduced via site-directed mutagenesis PCR and verified by sequencing.…”

Section: Methodsmentioning

confidence: 99%

ATP1A1-linked diseases require a malfunctioning protein product from one allele

Spontarelli

Young

Sweazey

et al. 2023

Preprint

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Heterozygous germline variants inATP1A1, the gene encoding the α1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT).ATP1A1variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αβ) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+and K+that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-nullATP1A1variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygousAtp1a1+/-knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found thatAtp1a1+/-mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of knownATP1A1-related diseases despite carrying a protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction byATP1A1variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

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Section: Methodsmentioning

confidence: 99%

ATP1A1-linked diseases require a malfunctioning protein product from one allele

Spontarelli

Young

Sweazey

et al. 2023

Preprint

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“…TCSPC experiments were performed as previously described. − TCSPC histograms for donors without the acceptor were acquired for permeabilized RyR2-expressing cells that were incubated for 15 min at RT in the presence of 50 nM FKBP12.6 labeled with Alexa Fluor 488 at positions 1, 6, 14, or 85. Subsequently, 1 μM Alexa Fluor 568-labeled ent -verticilide was added and incubated for 15 min, and TCSPC histograms were acquired for the donor in the presence of the acceptor.…”

Section: Methodsmentioning

confidence: 99%

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET

Šeflová,

Schwarz,

Smith

et al. 2023

ACS Chem. Biol.

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Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca 2+ -release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca 2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure−activity relationship studies, here, we used fluorescence lifetime (FLT)measurements of Forster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptorlabeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure−activity studies for lead optimization.

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“…Membrane protein enriched fractions were prepared from human heart tissue as previously described ( 55 ). Briefly, frozen heart tissue was placed in 5 ml of a solution containing 100 mM KCl, 2.5 mM K 2 HPO 4 , 2.5 mM KH 2 PO 4 , 2 mM EDTA, and protease inhibitors.…”

Section: Methodsmentioning

confidence: 99%

“…Membrane protein enriched fractions were prepared from transfected HEK cells as previously described ( 55 ). Fourty-eight hours post-transfection, HEK cells were scraped in 5 ml of homogenizing solution containing 250 mM sucrose, 10 mM Tris, and 2 mM EDTA pH 7.4 with protease inhibitors and centrifuged at 4000 g for 10 min at 4 °C.…”

Section: Methodsmentioning

confidence: 99%