Summary Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca2+ influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3’s pore-forming α1E subunit showed altered Ca2+ responses, reduced AE, and a strong sub-fertility phenotype. Surprisingly, AE depended on spatio-temporal information encoded by flux through CaV2.3—not merely the presence/ amplitude of Ca2+ waves. Using both studies in sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1’s lipid and sugar components and CaV2.3’s α1E and α2δ subunits. Our results provide mechanistic understanding of membrane lipid regulation of Ca2+ flux and therefore Ca2+-dependent cellular and developmental processes such as exocytosis and fertilization.

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It is well-established that syntaxin 1A (Sx1A), SNAP-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca(2+) channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while Sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca(2+)-independent coupling of the human neuronal P/Q-type calcium channel (Ca(V)2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of Ca(V)2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by Ca(V)1.2, Ca(V)2.2 and Ca(V)2.3. The distinct kinetic properties of Ca(V)2.1 acquired by close association with Syt1 and t-SNAREs, suggests that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca(2+). To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BAPTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted release mediated by Ca(V)2.1 is consistent with the model in which the VGCC plays a signaling role in triggering release, acting from within the exocytotic complex. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca(2+) entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential.

Diabetes is a high risk factor for dementia. High glucose may be a risk factor for dementia even among persons without diabetes, and in transgenic animals it has been shown to cause a potentiation of indices that are pre-symptomatic of Alzheimer's disease. To further elucidate the underlying mechanisms linking inflammatory events elicited in the brain during oxidative stress and diabetes, we monitored the activation of mitogen-activated kinsase (MAPKs), c-jun NH2-terminal kinase (JNK), p38 MAP kinases (p38MAPK), and extracellular activating kinsae1/2 (ERK1/2) and the anti-inflammatory effects of the thioredoxin mimetic (TxM) peptides, Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4) in the brain of male leptin-receptor-deficient Zucker diabetic fatty (ZDF) rats and human neuroblastoma SH-SY5Y cells. Daily i.p. injection of CB3 to ZDF rats inhibited the phosphorylation of JNK and p38MAPK, and prevented the expression of thioredoxin-interacting-protein (TXNIP/TBP-2) in ZDF rat brain. Although plasma glucose/insulin remained high, CB3 also increased the phosphorylation of AMP-ribose activating kinase (AMPK) and inhibited p70S6K kinase in the brain. Both CB3 and CB4 reversed apoptosis induced by inhibiting thioredoxin reductase as monitored by decreasing caspase 3 cleavage and PARP dissociation in SH-SY5Y cells. The decrease in JNK and p38MAPK activity in the absence of a change in plasma glucose implies a decrease in oxidative or neuroinflammatory stress in the ZDF rat brain. CB3 not only attenuated MAPK phosphorylation and activated AMPK in the brain, but it also diminished apoptotic markers, most likely acting via the MAPK–AMPK–mTOR pathway. These results were correlated with CB3 and CB4 inhibiting inflammation progression and protection from oxidative stress induced apoptosis in human neuronal cells. We suggest that by attenuating neuro-inflammatory processes in the brain Trx1 mimetic peptides could become beneficial for preventing neurological disorders associated with diabetes.

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.