The L-type Voltage-Gated Calcium Channel co-localizes with Syntaxin 1A in nano-clusters at the plasma membrane

Julia Sajman; Michael Trus; Daphne Atlas; Eilon Sherman

doi:10.1038/s41598-017-10588-4

The L-type Voltage-Gated Calcium Channel co-localizes with Syntaxin 1A in nano-clusters at the plasma membrane

Julia Sajman, Michael Trus, Daphne Atlas^*, Eilon Sherman

^*Corresponding author for this work

Racah Institute of Physics

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

The secretory signal elicited by membrane depolarization traverses from the Ca²⁺-bound α₁1.2 pore-forming subunit of the L-type Ca²⁺-channel (Cav1.2) to syntaxin 1 A (Sx1A) via an intra-membrane signaling mechanism. Here, we report the use of two-color Photo-Activated-Localization-Microscopy (PALM) to determine the relation between Cav1.2 and Sx1A in single-molecule detail. We observed nanoscale co-clusters of PAmCherry-tagged Sx1A and Dronpa-tagged α₁1.2 at a ~1:1 ratio. PAmCherry-tagged Sx1A^C145A, or PAmCherry-tagged Sx2, an inactive Cav1.2 modulator, in which Cys145 is a Ser residue, showed no co-clustering. These results are consistent with the crucial role of the single cytosolic Sx1ACys145 in clustering with Cav1.2. Cav1.2 and the functionally inactive transmembrane-domain double mutant Sx1A^C271V/C272V engendered clusters with a ~2:1 ratio. A higher extent of co-clustering, which coincides with compromised depolarization-evoked transmitter-release, was observed also by oxidation of Sx1ACys271 and Cys272. Our super-resolution-imaging results set the stage for studying co-clustering of the channel with other exocytotic proteins at a single-molecule level.

Original language	American English
Article number	11350
Journal	Scientific Reports
Volume	7
Issue number	1
DOIs	https://doi.org/10.1038/s41598-017-10588-4
State	Published - 1 Dec 2017

Bibliographical note

Funding Information:
The authors would like to thank David Sajman for his assistance in developing the co-clustering analyses. This research was supported by Grant no. 321993 from the Marie Skłodowska-Curie actions of the European Commission, the HU-HUJI fund for collaborative research, Grants no. 1417/13 and no. 1937/13 from the Israeli Science Foundation and for The H.L. Lauterbach Foundation.

Publisher Copyright:
© 2017 The Author(s).

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title = "The L-type Voltage-Gated Calcium Channel co-localizes with Syntaxin 1A in nano-clusters at the plasma membrane",

abstract = "The secretory signal elicited by membrane depolarization traverses from the Ca2+-bound α11.2 pore-forming subunit of the L-type Ca2+-channel (Cav1.2) to syntaxin 1 A (Sx1A) via an intra-membrane signaling mechanism. Here, we report the use of two-color Photo-Activated-Localization-Microscopy (PALM) to determine the relation between Cav1.2 and Sx1A in single-molecule detail. We observed nanoscale co-clusters of PAmCherry-tagged Sx1A and Dronpa-tagged α11.2 at a ~1:1 ratio. PAmCherry-tagged Sx1AC145A, or PAmCherry-tagged Sx2, an inactive Cav1.2 modulator, in which Cys145 is a Ser residue, showed no co-clustering. These results are consistent with the crucial role of the single cytosolic Sx1ACys145 in clustering with Cav1.2. Cav1.2 and the functionally inactive transmembrane-domain double mutant Sx1AC271V/C272V engendered clusters with a ~2:1 ratio. A higher extent of co-clustering, which coincides with compromised depolarization-evoked transmitter-release, was observed also by oxidation of Sx1ACys271 and Cys272. Our super-resolution-imaging results set the stage for studying co-clustering of the channel with other exocytotic proteins at a single-molecule level.",

author = "Julia Sajman and Michael Trus and Daphne Atlas and Eilon Sherman",

note = "Funding Information: The authors would like to thank David Sajman for his assistance in developing the co-clustering analyses. This research was supported by Grant no. 321993 from the Marie Sk{\l}odowska-Curie actions of the European Commission, the HU-HUJI fund for collaborative research, Grants no. 1417/13 and no. 1937/13 from the Israeli Science Foundation and for The H.L. Lauterbach Foundation. Publisher Copyright: {\textcopyright} 2017 The Author(s).",

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AU - Sherman, Eilon

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AB - The secretory signal elicited by membrane depolarization traverses from the Ca2+-bound α11.2 pore-forming subunit of the L-type Ca2+-channel (Cav1.2) to syntaxin 1 A (Sx1A) via an intra-membrane signaling mechanism. Here, we report the use of two-color Photo-Activated-Localization-Microscopy (PALM) to determine the relation between Cav1.2 and Sx1A in single-molecule detail. We observed nanoscale co-clusters of PAmCherry-tagged Sx1A and Dronpa-tagged α11.2 at a ~1:1 ratio. PAmCherry-tagged Sx1AC145A, or PAmCherry-tagged Sx2, an inactive Cav1.2 modulator, in which Cys145 is a Ser residue, showed no co-clustering. These results are consistent with the crucial role of the single cytosolic Sx1ACys145 in clustering with Cav1.2. Cav1.2 and the functionally inactive transmembrane-domain double mutant Sx1AC271V/C272V engendered clusters with a ~2:1 ratio. A higher extent of co-clustering, which coincides with compromised depolarization-evoked transmitter-release, was observed also by oxidation of Sx1ACys271 and Cys272. Our super-resolution-imaging results set the stage for studying co-clustering of the channel with other exocytotic proteins at a single-molecule level.

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The L-type Voltage-Gated Calcium Channel co-localizes with Syntaxin 1A in nano-clusters at the plasma membrane

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