Depolarization of vertebrate skeletal muscle causes intracellular calcium release, an event depending on specialized junctions between the plasma membrane (PM) and sarcoplasmic reticulum (SR). It is known that depolarization is ‘‘sensed’’ by a voltage-gated calcium channel located in the PM and containing CaV1.1 as its principal subunit, that calcium is released from the SR via RyR1, and that this does not require external Ca2þ entry. Thus, it is thought that activation of RyR1 is driven by depolarizationinduced conformational changes in CaV1.1 which are mechanically transmitted to RyR1, either directly or via intervening proteins. Based on muscle gene knockouts, at least two additional proteins are required for this conformational coupling, but knockouts cannot reveal whether yet-to-be identified proteins, or sets of proteins with overlapping functions, are also critical. Thus, we used reconstitution in tsA201 cells to identify a minimal set of proteins required for conformational coupling. We found that expression of junctophilin2 was effective at promoting ER-PM junctions, and that CaV1.1 and RyR1 both targeted to these junctions in the presence of the CaV1.1 auxiliary subunit b1a. Moreover, tsA201 cells expressing these four proteins, and the adapter protein Stac3, produced depolarization-triggered Ca2þ transients which were independent of extracellular Ca2þ entry and which increased in amplitude as a saturating function of voltage, as expected for conformational coupling. Additionally, freeze-fracture electron microscopy indicated that CaV1.1 and RyR1 were physically linked in these cells, thus establishing that the five expressed proteins are sufficient for conformational coupling. The ability to reconstitute conformational coupling with a minimal set of proteins may provide a system for obtaining high resolutions structures of CaV1.1 and RyR1 as part of a functioning complex, which will be necessary for understanding the molecular mechanism of depolarization-evoked calcium release in skeletal muscle.

Perni, S., Lavorato, M., Beam, K.G. (2018). De Novo Reconstitution of Skeletal Muscle Voltage-Induced Calcium Release. BIOPHYSICAL JOURNAL, 114(3), 40A-40A.

De Novo Reconstitution of Skeletal Muscle Voltage-Induced Calcium Release

Perni, S.;
2018

Abstract

Depolarization of vertebrate skeletal muscle causes intracellular calcium release, an event depending on specialized junctions between the plasma membrane (PM) and sarcoplasmic reticulum (SR). It is known that depolarization is ‘‘sensed’’ by a voltage-gated calcium channel located in the PM and containing CaV1.1 as its principal subunit, that calcium is released from the SR via RyR1, and that this does not require external Ca2þ entry. Thus, it is thought that activation of RyR1 is driven by depolarizationinduced conformational changes in CaV1.1 which are mechanically transmitted to RyR1, either directly or via intervening proteins. Based on muscle gene knockouts, at least two additional proteins are required for this conformational coupling, but knockouts cannot reveal whether yet-to-be identified proteins, or sets of proteins with overlapping functions, are also critical. Thus, we used reconstitution in tsA201 cells to identify a minimal set of proteins required for conformational coupling. We found that expression of junctophilin2 was effective at promoting ER-PM junctions, and that CaV1.1 and RyR1 both targeted to these junctions in the presence of the CaV1.1 auxiliary subunit b1a. Moreover, tsA201 cells expressing these four proteins, and the adapter protein Stac3, produced depolarization-triggered Ca2þ transients which were independent of extracellular Ca2þ entry and which increased in amplitude as a saturating function of voltage, as expected for conformational coupling. Additionally, freeze-fracture electron microscopy indicated that CaV1.1 and RyR1 were physically linked in these cells, thus establishing that the five expressed proteins are sufficient for conformational coupling. The ability to reconstitute conformational coupling with a minimal set of proteins may provide a system for obtaining high resolutions structures of CaV1.1 and RyR1 as part of a functioning complex, which will be necessary for understanding the molecular mechanism of depolarization-evoked calcium release in skeletal muscle.
Perni, S., Lavorato, M., Beam, K.G. (2018). De Novo Reconstitution of Skeletal Muscle Voltage-Induced Calcium Release. BIOPHYSICAL JOURNAL, 114(3), 40A-40A.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11365/1216064