Intra-membrane Signaling Between the Voltage-Gated Ca2+-Channel and Cysteine Residues of Syntaxin 1A Coordinates Synchronous Release

Abstract

The interaction of syntaxin 1A (Sx1A) with voltage-gated calcium channels (VGCC) is required for depolarization-evoked release. However, it is unclear how the signal is transferred from the channel to the exocytotic machinery and whether assembly of Sx1A and the calcium channel is conformationally linked to triggering synchronous release. Here we demonstrate that depolarization-evoked catecholamine release was decreased in chromaffin cells infected with semliki forest viral vectors encoding Sx1A mutants, Sx1AC271V, or Sx1AC272V, or by direct oxidation of these Sx1A transmembrane (TM) cysteine residues. Mutating or oxidizing these highly conserved Sx1A Cys271 and Cys272 equally disrupted the Sx1A interaction with the channel. The results highlight the functional link between the VGCC and the exocytotic machinery, and attribute the redox sensitivity of the release process to the Sx1A TM C271 and C272. This unique intra-membrane signal-transduction pathway enables fast signaling, and triggers synchronous release by conformational-coupling of the channel with Sx1A.

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

“…The oxidation by auranofin can be reversed by exposure to reducing conditions e.g thiol-reagents such as N-acetylcysteine amide (NAC-amide; AD4) 39 or thioredoxin-mimetic peptides TXM-CB3 18 , 37 . Following exposure to AuF, the cells were washed, and then pre-incubated for additional 30 min with 1mM AD4 (Fig.…”

“…The functional coupling between the channel and Sx1A is essential for triggering transmitter release, and requires the presence of the two highly conserved TMD Sx1ACys271 and Cys272 18 . The two-color PALM imaging analysis revealed ~2:1 ratio nano-clusters of Sx1A CC/VV /α 1 1.2, compared to the ~1:1 ratio observed in nano clusters of wt Sx1A/α 1 1.2.…”

“…Similarly, the Sx1A double mutant Sx1A C271V/C272V does not modify current amplitude, and does not support depolarization-evoked secretion in a reconstituted system 12 . The Sx1A C271V/C272V mutant operates also as a dominant negative in depolarization-induced catecholamine release in chromaffin cells 18 . The interaction of Sx1A TMD with the channel led to the proposal that a signal induced by conformational change during membrane depolarization is transmitted via Sx1A TMD, triggering transmitter release within microseconds 18 .…”

SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting

“…There is precedence for syntaxins to bind to non-SNARE partners involved in vesicular transport, including vesicular cargoes such as vesicle coat proteins, tethering factors, and Rab GTPases thought to be involved in regulating the targeting and docking of vesicles [ 43 , 44 ]. In addition, some syntaxins have been shown to bind proteins not involved in vesicular transport such as ion channels, for example Syntaxin 1A binds to multiple subtypes of calcium channels in the plasma membrane, an interaction required for voltage sensitive neurotransmitter release [ 45 , 46 ]. It is thus possible that direct binding between RDS and Syn3B/SNAP-25 means RDS may play a role in the actual fusion event or determination of tethering with the targeted membrane.…”

Specific Ion Channels Control Sensory Gain, Sensitivity, and Kinetics in a Tonic Thermonociceptor

“…Specific VGCCs Are Required to Maintain FLP Sensory Gain and Thermal Sensitivity VGCCs are major actors in the control of calcium influx from the extracellular space and play multiple roles from signal amplification and transmission to vesicle exocytosis (Bachnoff et al, 2013). The C. elegans genome contains single genes encoding each of three VGCC pore-forming subunit classes: egl-19 animals quantified with two protocols as schematically depicted: direct stimulation at 34 C from a 20 C baseline versus indirect stimulation to 34 C with an intermediate step at 28 C. (A) Average calcium traces (lines) with SEM as gray shades.…”