Figure 3. Effect of point mutations in ligand gating.

The possible effects of point mutations in the LBD gating of AtGLR3.2 were assayed by the transfection of mammalian COS-7 cells expressing a Ca²⁺ indicator (YC3.6). A, Expression of wild-type channel alone, shows its Ca²⁺ conductance to be gated by Glycine (Gly) at 1.0 mM. The experimental sequence is shown on the top black/yellows bar. Cells are Ca²⁺-starved with EGTA and then perfused with 14.5 mM Ca²⁺. In the absence of ligand (black dots) a slight increase occurs in cytosolic Ca²⁺. When the experiment is done in the presence 0.5 mM Gly, this elevation is slightly, but significantly, prolonged (p<0.01), but in the presence of 1.0 mM Gly there is a visible and statistically significant elevation of cytosolic Ca²⁺ (p<10⁻⁶ to control and p=0.01 to 0.5 mM). B, Simultaneous expression of AtGLR3.2 and AtCNIH4 renders the channel gated by both Met (red) and Gly (green) at 0.5 mM in comparison to the control (p<0.01 for all comparisons). However, when the critical residue 133 is substituted from Arginine to Alanine (C) the channel behaves as being constitutively open (black; compare with black control in B). Data are represented as mean ± SEM. All statistics obtained by two-way ANOVA with TukeyHSD.

See also Figures S3 and S4.