Neurobio Test2: Recitation 5 Flashcards
Main question?
Is synaptotagmin I the calcium sensor invovled in NT release?
Explain the structure of the Synaptotagmin 1
Top loops bind calcium. CA2 domain ligates several calcium ions in incomplete coordination spheres formed primarily by aspartate residues.
Ca2+ binding to synaptotagmin I may regulate exocytosis in vivo. How the authors proved this hypothesis?
Point mutation that reduced calcium affinity w/o changing 3D structure. Introduced mutation into endogenous synaptotagmin I gene of mice and observed decrease in Ca2+ mediated NT release.
Mutations in the positively charged residues surrounding the Ca2+-binding sites of the C2A domain of synaptotagmin I influence Ca2+ binding. How the authors test it and which are the results?
Characterized mutations by NMR spec. Mutations caused little perturbations in spectra, indicating minimal effect on 3D structure.
Explain different results observed between R233Q and K236Q point mutations.
Phenotype of R233Q (reduced Ca2+ triggered NT release) not due to nonspecific electrostatic changes since K236Q did not have this effect. Indicates synaptotagmin I is at least involved in Ca2+ sensing related to NT release.
Explain different alternatives that could account for the Ca2+ sensor phenotype observed in the R233Q mutation.
- Ca2+ influx is inhibited
- Ca2+ buffering is increased
- R233 mediates binding of an unidentified protein to synaptotagmin I which is the ‘true’ Ca2+ sensor
Disprove different alternatives that could account for the Ca2+ sensor phenotype observed in the R233Q mutation.
- Ca2+ influx inhib: Ca2+ channel did not change
- Calcium buffering increased: buffering needed for effect higher than expected for the amount of calcium bound by synaptotagmin
- mediates binding of unidentified protein: this protein would have to interact in the same way with synaptotagmin bound and not bound to calcium, or synaptotagmin is a calcium sensor in release after all
What is the mechanism of action of synaptotagmin I as a Ca2+ sensor/transducer in release?
Present on docked vesicles at active zone. Interacts with syntaxin, which plays role in vesicle fusion. Syntaxin interaction needs less caclium to happen for wild type synaptotgamin I.
How the authors introduced the point mutations in the synaptotagmin 1 gene? Explain Knock-in technique.
Targeting vectors. To ensure that all analysis was performed on precisely matched controls, all analyses were performed on littermates derived from matings between double heterozygotes. Knockins had a substitution.
Main discovery
Synaptotagmin I as a sensor for Ca2+ in triggering neurotransmitter release to initiate signal transmission
Breifly outline the structure of synaptotagmin I.
Two C2 domains composed of eight-stranded beta-sandwiches. Three flexible loops on top, four at bottom. Loops 1 and 3 of C2A each bind three calcium ions.
What contributes to an increased Ca2+ binding affinity by Synaptotagmin I and how?
Positively charged arginine residue binds phospholips with negatively charged head groups, which in turn bind calcium. Ternary complex of phospholipid headgroups + two loops binds calcium.
What are some of the challenges posed in elucidating the specific function of Synaptotagmin I in binding Ca2+ aiding to neurotransmitter release? How did the authors circumvent this?
Don’t want to alter a single C2 domain since the domains might cooperate. Don’t want to alter 3D structure. Point-mutation that alters calcium affinity w/o changing 3D structure.
How did the authors contend that R233Q point mutation decreases the overall Ca2+ affinity of the C2A domain?
Tested calcium-dependent phospholipid binding by adding liposomes with negatively charged head groups.
How does R223Q mutation selectively impair neurotransmitter release (in vivo)?
Amplitude of EPSC lower in mutant, indicating calcium mediated NT release does not work as well. Mutants also showed facilitation - response got bigger with time because more and more calcium built up, showing the calcium binding does not work as well.
