Tryptophan replacement experiment reveals the importance of syt-membrane interaction in vesicular

release efficiency and the cooperation of the two C₂ domains in fulfilling the functions of syt

4. Discussion

There are three classes of interactions within syt, which have been found to determine/affect the functions of syt in vitro: A) the five aspartates in each C2

domain functioning as the essential domains for Ca²⁺ mediated phospholipid binding; B) the basic residues distributed in the top loops or flanking side of syt that interacts with multiple molecules; C) the hydrophobic residues in the loop region, thought to undergo Ca²⁺ dependent insertion into the phospholipid membrane (Chapman ER and Davis AF, 1998; Bai J, et al., 2002). By replacing these hydrophobic residues with the most hydrophobic amino acid, tryptophan, ionic mediated protein-syt interaction in these regions should be unlikely affected, but the Ca²⁺ dependent phospholipid binding will be enhanced.

Previous analysis was performed by Ok-Ho Shin to determine the Ca²⁺

dependent syt-phospholipid binding of these tryptophan mutants in vitro (Fig.

4.1). As predicted by the design, a gradual increase of the Ca²⁺ dependent phospholipid binding was found in these three mutants. This suggests that the tryptophan mutation can drastically enhance the Ca²⁺ dependent phospholipid binding ability of syt. Consistent with the biochemical finding, my data showed enhanced synaptic release efficiency in all three tryptophan mutants. The nice correlation between biochemical and electrophysiological findings further reinforces the idea that syt-phospholipid interaction is crucial for syt to induce vesicular release.

Figure 4.1. Ca²⁺ dependent phospholipid binding of different syt tryptophan mutants comparison with the sytWT (data provided by Ok-Ho Shin, for detailed methodological explanation, see Shin OH, et al., 2002.)

4. Discussion

The three fold increase in the apparent Ca²⁺ sensitivity in sytC2AB6W mutant leads to another interesting question: how is the EPSC time course of this mutant comparing to sytWT rescue? The idea behind the question is based on the previous model that evoked asynchronous release is regulated by another Ca²⁺

sensor with higher Ca²⁺ affinity (Goda Y and Stevens CF, 1994). If a higher affinity Ca²⁺ sensor for asynchronous release exists, we may expect a certain change (i. e. increase in asynchronous release) in the evoked release time course in the neurons rescued by “super syt” (sytC2AB6W). The decovolution of evoked EPSC time course between the sytWT and the sytC2AB6W rescued neurons overlaps. A more detailed analysis of the time constants of synchronous and asynchronous release and the contribution of each component to total release reveals no change between the sytWT and the sytC2AB6W rescued neurons as well (Fig 4.2). These data suggest that the Ca²⁺ sensitivity of the asynchronous release Ca²⁺ sensor may be more than three-fold higher than that of syt. Alternatively, the explanation can be that syt solely plays a role in regulating synchronous vesicular release.

Figure 4.2 The high Ca²⁺ sensitivity mutant does not yield significant changes in the time course of neurotransmitter release. A. Top, exemplary EPSCs from SytWT (black) and SytC₂AB6W rescues (red); Below, EPSCs deconvolved with mean mEPSC time course to reveal the vesicular release rate. B. Release time course were fitted by two exponential equation. Bar plot of mean time constants (left) and relative amplitude of the fast component of release (right). Student’s t-test shows no difference between sytWT (black) and

4. Discussion

The finding that the sytC2A3W and the sytC2B3W both display a similar behavior in the short term depression and an increase of apparent Ca²⁺ sensitivity indicates the important function of the C2A domain in synaptic transmission.

Previous studies of syt in Drosophila suggested that the C2A domain is not important for synaptic function (Mackler JM, et al., 2002). However other studies implicated the Ca²⁺ binding region of the C2A domain as part of the Ca²⁺ senor molecule (Stevens CF and Sullivan JM, 2003). Here, our study clearly shows that the C2A domain is important for syt function and that both C2A and C2B domains contribute almost equally to regulate the Ca²⁺ sensitivity and probability of vesicle release.

Another finding is the gradual increase of mEPSC frequency in the order of sytWT< sytC2A3W< sytC2B3W< sytC2AB6W. This observation can be correlated to the gradual increase of the apparent Ca²⁺ sensitivity in the three tryptophan mutants, since the elevation of the Ca²⁺ sensing ability of these mutants may let the vesicles start releasing in residue Ca²⁺ level. To test this hypothesis, the rescued neurons (with sytWT or sytC2AB6W) were incubated in 50 µM EGTA-AM for 15 min (37 ºC) in order to accumulate a high concentration of EGTA in the presynaptic terminals, and then mEPSCs were recorded in presence of 300 nM TTX. To our surprise, Student’s t-test indicated no change of spontaneous release rate either in the sytWT or in the sytC2AB6W group after EGTA-AM application (The mEPSC frequency after EGTA-AM application was normalized to the mEPSC frequency before EGTA-AM application, sytWT 93.8± 12.5%, n=17; sytC2AB6W 81.1± 16.2%, n=19). The unchanged mEPSC frequency suggests that the spontaneous release rate is Ca²⁺ independent. Another experiment recording mEPSC of sytC2AB6W rescued neurons in the presence of 0 mM Ca²⁺ and 4 mM Ca²⁺ was performed by Dr. Rhee JS, to further test this idea. In this experiment, the spontaneous release rate was unchanged in either condition (data not shown), which also support the previous conclusion that the spontaneous release rate is Ca²⁺ independent in sytC2AB6W. In summary, the increased mEPSC frequency in sytC2AB6W mutant is not due to its three-fold

4. Discussion

increase in the apparent Ca²⁺ sensitivity compared to sytWT. Possible explanation can be that the sytC2AB6W mutant may enhance Ca²⁺ independent phospholipid interaction thus to increase spontaneous vesicle fusion rate.

4.2.3 Asymmetrical distribution of basic residues for regulation