This observation could be due to a difference in the fusion
Metformin concentration mechanism for TMR- versus lipid-anchored syntaxin-1A, so that the distance of the SNARE motif to the membrane anchor is functionally irrelevant for the latter. Alternatively, this finding could be due to a different optimal distance of the SNARE motif from the membrane anchor for TMR- and lipid-anchored syntaxin-1. To differentiate between these two possibilities and to test whether lipid-anchored and wild-type syntaxin-1A act by similar mechanisms, we examined the effect of further amino acid insertions between the SNARE motif and the lipid anchor in syntaxin-1A. In these experiments, we tested insertions of additional 3, 7, or 14 residues
on top of the seven-residue insertion characterized above (referred to as Syntaxin-1AΔTMR+10i, Syntaxin-1AΔTMR+14i, and Syntaxin-1AΔTMR+21i, respectively; Figure S4A). We found that all insertion mutants of lipid-anchored syntaxin-1A rescued the impairment of spontaneous release in syntaxin-deficient neurons (Figures 3A and 3B). Unexpectedly, the longer insertions seemed to even increase mIPSCs, suggesting that they may “unclamp” spontaneous release. Sunitinib manufacturer We detected no consistent change in the amplitudes and kinetics of spontaneous many release under any condition (Figure S4B). When we examined action-potential-evoked release, however, we observed that similar to TMR-anchored syntaxin-1A, insertion of an additional three amino acids in
lipid-anchored synaxin-1A on top of the seven-residue insertion (which by itself improved evoked release; Figure 2) blocked evoked release (Figure 3C). This phenotype was associated with a large increase in the desynchronization of release as measured via the variability of rise times (Figure 3D). Moreover, the additional insertions into lipid-anchored syntaxin-1A also blocked the ability of syntaxin-1A to rescue fusion induced by stimulus trains in syntaxin-deficient neurons (Figure 3E). Thus, lipid-anchored syntaxin-1A essential behaves like wild-type syntaxin-1A, with the same selective requirement for a precise distance between the SNARE motif and the membrane anchor for evoked but not for spontaneous release, except that the optimal distance of the SNARE motif from the membrane anchor appears to be slightly longer. Most studies demonstrating an essential role for a SNARE TMR in fusion were performed with synaptobrevin-2.