The differences
between syb2 and vti1a slope values during chemical stimulation are significant in both 2 and 8 mM extracellular Ca2+. Figures 4C and 4F depict the cumulative data as a percentage of total internal fluorescence after NH4Cl application. The inset depicts a sample graph indicating how the changes http://www.selleckchem.com/products/NVP-AUY922.html in fluorescence were calculated. The percentage of vti1a residing in internal compartments released during 90 mM K+ stimulation is significantly less than that of syb2 in both 2 and 8 mM CaCl2. The percentage of vti1a molecules that are trafficked at rest is significantly more than syb2 in the presence of 2 mM extracellular Ca2+, but the spontaneous trafficking of syb2 and vti1a is approximately equal in the presence of elevated extracellular Ca2+, corroborating earlier results (Figure 2). These results show that the majority of vti1a trafficking occurs at rest, and even with strong elevated K+ stimulation, vti1a-containing vesicles are released at a lower rate compared to those containing agonist syb2. Furthermore, the simultaneous visualization
of syb2 and vti1a trafficking during the 90 mM K+ stimulation strongly suggests that these proteins largely reside in different vesicular pools. We next assessed the simultaneous trafficking of VAMP7-pHluorin and syb2-mOrange. In experiments like those described for vti1a, syb2-mOrange was expressed in the same neurons as VAMP7-pHluorin, and their spontaneous and evoked trafficking was measured simultaneously. Syb2-mOrange trafficked robustly both at rest and with 90 mM K+ stimulation, but significantly less VAMP7 trafficking was observed under either condition (Figure S5). These results are comparable to earlier findings (Figure 2). While we did not observe robust mobilization of VAMP7-pHluorin either at rest or with stimulation, old in contrast to Hua et al. (2011), this is likely a
result of the autoinhibitory actions of the longin domain, which was present in our full-length VAMP7 construct. Still, a measurable amount of VAMP7-pHluorin trafficking was seen in the same synapses as syb2-mOrange, which agrees well with the basic finding of their report that VAMP7 is targeted to a subpool of SVs. In light of recent work showing a role of endosomal sorting in SV recycling (Hoopmann et al., 2010), we evaluated the overlap between the trafficking behaviors of vti1a and two bona fide endosomal markers, transferrin receptor (TfR) (Kennedy et al., 2010) and syntaxin-6 (Rizzoli et al., 2006) (Figure S6). TfR and syntaxin-6 showed limited spontaneous trafficking in synapses compared to vti1a. Thus, although vti1a resides in endosomes (Antonin et al., 2000a, Bethani et al., 2009 and Kunwar et al., 2011) in addition to its presence on SVs (Antonin et al., 2000b and Takamori et al.