Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells

Hu, Yifan; Iyer, Kavita A.; Nayak, Ashok R.; Kurebayashi, Nagomi; Murayama, Takashi; Samsó, Montserrat

doi:10.21769/bioprotoc.4112

Cited by 4 publications

(3 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, recombinant homotetrameric rabbit RyR1 Y523S was expressed stably in HEK293 cells using the Flp-In T-REx system and a doxycycline-inducible promoter as previously reported ( 18 , 19 ). RyR1s were purified using sucrose density gradient centrifugation ( 20 ), supplemented with FKBP12.6, and reconstituted into nanodiscs under either closed-state (2 mM ethyleneglycol- bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid; EGTA, i.e., submicromolar free Ca 2+ ) or open-state (1.5 µM free Ca 2+ , 5 mM adenosine 5′-triphosphate; ATP) conditions ( SI Appendix , Table S1 ). Cryo-EM yielded 3D reconstructions of the mutant RyR1 at global resolutions of 4.0 and 4.1 Å, for the closed- and open-state conditions, respectively ( SI Appendix , Figs.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Iyer

Klose

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Ryanodine receptors (RyRs) are main regulators of intracellular Ca 2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a “spacer” within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca 2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca 2+ , increasing by ∼20-fold the potency of Ca 2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Purification of recombinant RyR1 Y523S from HEK293 cells was performed in accordance with previously published protocol ( 17 , 20 ).…”

Section: Methodsmentioning

confidence: 99%

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Iyer

Klose

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Microsomes were purified from rabbit back and hind leg muscles through differential centrifugation as previously described (Hu et al, 2021, Samsó et al, 2009. 100 mg frozen membranes were thawed and solubilized in buffer A containing 20 mM MOPS pH 7.4, 1 M NaCl, 9.2% (w/v) CHAPS, 2.3% (w/v) Phosphatdylcholine (PC; Sigma), 2 mM DTT and protease inhibitor cocktail for 15 mins at 4 o C. The solubilized membranes were centrifuged at 100,000 x g for 60 min and the pellet was discarded.…”

Section: Purification Of Ryr1 From Rabbit Skeletal Muscle and Reconst...mentioning

confidence: 99%

Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM

Nayak

Samsó

2022

eLife

View full text Add to dashboard Cite

Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryo-EM of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca2+ conditions revealed an open and a closed-inactivated conformation. Ca2+ binding to the high affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca2+-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly-knit subunit interface contributed by a fully occupied Ca2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca2+ activation prerequisite for Ca2+ inactivation and provides for seamless transition from inactivated to closed conformations.

show abstract

Catching the next wave of recombinant RyR2 cryo-EM structures. Commentary on “Molecular basis for gating of cardiac ryanodine receptor explains the mechanisms for gain- and loss-of function mutations”

Miotto

Marks²

2022

Cell Calcium

View full text Add to dashboard Cite

Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells

Cited by 4 publications

References 8 publications

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM

Catching the next wave of recombinant RyR2 cryo-EM structures. Commentary on “Molecular basis for gating of cardiac ryanodine receptor explains the mechanisms for gain- and loss-of function mutations”

Contact Info

Product

Resources

About