Figure 7. Retinal ribbon specific STX3B has a glutamate at position AA 259 rendering its JMD similar to that of STX1AK260E and E259K mutation on STX3B acts as a molecular on-switch.

(A) Position of the AA 259 on STX3B’s JMD. (B) Example image of SDS-PAGE of the electrophoretic analysis of lysates obtained from STX1-null neurons transduced with STX1A^WT, STX3B^WT, or STX3B^E259K. (C) Example images of immunofluorescence labeling for Bassoon and STX3B, shown as magenta and cyan, respectively, in the corresponding composite pseudocolored images obtained from high-density cultures of STX1-null hippocampal neurons rescued with STX1A^WT, STX3B^WT, or STX3B^E259K. Scale bar: 10 µm (F) Quantification of the immunofluorescence intensity of STX3B as normalized to the immunofluorescence intensity of Bassoon in the same ROIs as shown in (C). The values were then normalized to the values obtained from STX3B^WT neurons. (E) Example traces (left) and quantification of the amplitude (right) of excitatory postsynaptic currents (EPSCs) obtained from hippocampal autaptic STX1-null neurons rescued with STX1A^WT, STX3B^WT, or STX3B^E259K. (F) Quantification of the decay time (80–20%) of the EPSC recorded from the same neurons as in (E). (G) Example traces (left) and quantification of readily releasable pool (RRP) recorded from the same neurons as in (E). (H) Quantification of vesicular probability (Pvr) recorded from the same neurons as in (E). (I) Example traces (left) and quantification of the frequency (right) of miniature excitatory postsynaptic currents (mEPSCs) recorded from the same neurons as in (E). Data information: The artifacts are blanked in example traces in (E and G). The example traces in (I) were filtered at 1 kHz. In (D–I), data points represent single observations, the violin bars represent the distribution of the data with lines showing the median and the quartiles. Red and blue annotations (stars and ns) on the graphs show the significance comparisons to STX1A^WT and STX3B^WT, respectively. Non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test was applied to data in (C–F); ***p≤0.001, ****p≤0.0001. The numerical values are summarized in source data.