To investigate whether Rab3A could mediate vesicle binding to the synaptic ribbon, we expressed in E. coli
a recombinant version of Rab3A, which after purification was tagged with a small-molecule fluorophore (). We deleted the normal C-terminus of Rab3A, removing the lipidation motif required for vesicle membrane anchoring (Johnston et al., 1991
). The Rab3A construct was also modified on the N-terminus to include a Q-tag labeling domain (PKPQQFM), which enabled enzymatic conjugation to a cadaverine-containing fluorophore derivative (Lin and Ting 2006
). Finally, a His-tag was added to the N-terminus for affinity purification on a Ni2+
column. Polyacrylamide gel electrophoresis showed a single major band both before and after fluorophore labeling (). The purified protein, which we named Fl-Rab3A-Δ, was soluble up to at least 400 μM in physiological buffer.
Fl-Rab3A-Δ binds to synaptic ribbons
The purified Fl-Rab3A-Δ protein was introduced through a patch pipette into giant rod and cone photoreceptors of the tiger salamander. Fl-Rab3A-Δ accumulated in spots at the base of the synaptic terminals of these cells, co-localizing with structures labeled with Alexa-488-tagged RIBEYE binding peptide (Alexa-488-RBP) (), a marker of the synaptic ribbon(Zenisek et al., 2004
). Co-localization of the two proteins was observed in each of the 109 presumptive ribbons in a total of 22 rods examined. Selective excitation of Fl-Rab3A-Δ resulted in photobleaching without affecting Alexa-488-RBP fluorescence (). The independent photobleaching confirms that co-localization of the two proteins was not an artifact of spectral overlap between their fluorophores, but rather represents binding to a common structure. Since RBP binds specifically to synaptic ribbons (Zenisek et al., 2004
), these findings strongly suggest that Rab3A also binds to the ribbons.
We estimated the density of Rab3A binding sites on the ribbon by determining the amount of Fl-Rab3A-Δ that is required to saturate labeling (). Using the fluorescence intensity of the pipette as a reference for dye concentration, we estimated the density to be 3077 ± 352 molecules/μm2
(n=5), far exceeding the number of vesicles on a fully packed synaptic ribbon (~400/μm2
) (Thoreson et al., 2004
). This indicates that there are sufficient binding sites to accommodate at least one Rab3A from every tethered vesicle.
Rab3A cycles between active GTP-bound and inactive GDP-bound forms, but only the GTP-bound form binds to RIM (Geppert and Südhof, 1998
). We found that ribbon labeling by Fl-Rab3A-Δ occurred when the pipette contained GTP, but not GDP-β-S, a non-phosphorylatable GDP analogue (). Rab3A can be locked into a GTP-bound active configuration by mutating the GTPase active site (the Q81L mutation; Brondyk et al., 1993
). GTPase assays confirmed that both the unlabeled and labeled forms of Rab3A-ΔQ81L had <20% of the activity of the wild-type protein (). Introducing the Q81L mutation into Fl-Rab3A-Δ did not disrupt the initial binding to ribbons (Fspot
of mutant=2.4 vs. wild-type=2.7, P
>0.05), but it dramatically reduced the turnover of Rab3A binding, as evidenced by a decrease in Fluorescence Recovery After Photobleaching (FRAP) on the ribbon as compared to the wild-type protein (mean FRAP=55% for wild-type vs. 19% for the Q81L mutant; ). The slower dissociation rate of the Q81L mutant results in the protein having a higher apparent affinity for the synaptic ribbon (). FRAP of wild-type and mutant protein in small cytoplasmic regions was fast (<0.1 sec) indicating that both proteins are freely diffusible and have a high mobility when they are unbound. These results indicate that GTPase activity is necessary for bound Rab3A to exchange with free Rab3A in the cytoplasm, suggesting that binding and dissociation is mediated by a GTP-GDP cycle. Incomplete FRAP on ribbons labeled with wild-type Rab3A-Δ may result from photodamage of the ribbon machinery, as shown previously with fluorophore-assisted light inactivation via labeled RBP (Snellman et al., 2011
Fl-Rab3A-Δ binding to ribbons is GTP-dependent
To determine whether Rab3A binding is essential for synaptic transmission, we obtained paired recordings from a presynaptic photoreceptor and a postsynaptic OFF bipolar cell in a retinal slice. Photoreceptors were depolarized from −70 to −10 mV to open voltage-gated Ca2+
channels and trigger enough Ca2+
-dependent exocytosis to empty the readily-releasable pool (Rabl et al., 2006
). The depolarizing stimulus was repeated at 30 sec intervals, sufficient for complete refilling of the pool. Over 10 min of recording, cone-driven EPSCs decreased little when wild-type Rab3A-Δ or no protein at all was included in the patch pipette (<10%) (). However, when Rab3A-ΔQ81L was included in the pipette the EPSCs diminished by ~67% within 10 min. The voltage-gated Ca2+
current was unaffected by Rab3A-ΔQ81L. Hence Rab3A-ΔQ81L must suppress some step in the synaptic vesicle delivery or release process.
Rab3A-ΔQ81L blocks synaptic release from rods and cones
Rod-driven EPSCs had a fast component (f-EPSC) similar to cone-driven EPSCs, but also a secondary slower component (s-EPSC) (). Inclusion of Rab3A-ΔQ81L in the rod patch pipette reduced the f-EPSC by ~59%, but had no effect on the s-EPSC (). The f-EPSC appears to be mediated by a direct monosynaptic connection from the rod to the bipolar cell whereas the s-EPSC is mediated by electrically coupled rods with synaptic contacts onto the same bipolar cell. Because the Rab3A-ΔQ81L cannot cross gap junctions, it reduces synaptic release only from the rod dialyzed by the patch pipette, hence selectively reducing the f-EPSC. Consistent with this explanation, the gap junction uncoupler carbenoxolone had little effect on the f-EPSC, but it blocked the s-EPSC (), consistent with previous observations (Cadetti et al., 2005
If Rab3A binding to the ribbon is a necessary step in synaptic release, exogenous Rab3A-Δ should compete with endogenous Rab3A in inhibiting release. Furthermore, Rab3A binding sites should be more frequently vacated when the vesicle release rate is faster, because there will be more opportunities for Rab3A to bind. We tested these predictions by varying the stimulation frequency during Rab3A-ΔQ81L dialysis into rods. With a 10 sec interval between depolarizing stimuli, EPSCs were suppressed about twice as fast as with a 30 sec interval (). Hence while Rab3A-ΔQ81L has little effect on vesicles that are already docked and primed for release, it suppresses the delivery of new vesicles into the immediately releasable pool.
Rab3A binding sites are necessary for synaptic vesicle replenishment