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Sci Rep. 2017 Sep 12;7(1):11350. 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.

Author information

1
Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel.
2
Dept. of Biological Chemistry, Institute of Life Sciences, Jerusalem, 91904, Israel.
3
Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. daphne.atlas@mail.huji.ac.il.
4
Dept. of Biological Chemistry, Institute of Life Sciences, Jerusalem, 91904, Israel. daphne.atlas@mail.huji.ac.il.
5
Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. sherman@phys.huji.ac.il.

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.

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