Figure 1. N-cadherin sJMD regulation of HVA Ca²⁺ current requires binding to p120-catenin and RhoA activation.

A) Whole-cell voltage-clamp recordings of isolated inward Ca²⁺ currents from acutely dissociated ciliary ganglion neurons. Representative current response traces (upper panel) were elicited by 100 ms duration 10 mV steps in the voltage holding potential sequentially from −50mV to 50mV (lower panel). B) Current density plot shows averaged peak current reduction associated with infusion of N-cadherin sJMD as compared with control solution (control circles 35.4 ± 1.6, n=21; sJMD (1μM) open triangles 20.4 ± 1.8, n=12). Application of sJMD780AAA has no effect on current density (sJMD 780AAA (1μM) squares 38.4 ± 2.6, n=8). Simultaneous application of sJMD and C3 exotransferase substantially reverts peak current reduction associated with sJMD (sJMD (1μM) + C3 (0.5μM) open diamonds 32.5 ± 2.7, n=6). C) GST-tagged sJMD and GST-sJMD-780AAA were incubated with recombinant 6XHis-tagged p120-catenin (p120-228). Protein complexes were pulled down with glutathione-coated agarose beads, and analyzed by Western bolt with anti-His antibodies to detect p120-catenin. Lane 1, 5 μg of recombinant p120-228 runs at its predicted size of 89kDa. p120-catenin pulled down by recombinant GST (lane 2), sJMD-780AAA (lane 3), and sJMD (lane 4). Mutation of the p120-catenin binding site (sJMD-780AAA) substantially reduces p120-catenin binding to sJMD. D) Application of a dominant-active form of RhoA (DA-RhoA) reduces peak Ca²⁺ currents amplitude (DA-RhoA (0.4μM) open squares 19.8 ± 2.7, n=8). Simultaneous application of DA-RhoA and sJMD shows that their inhibitory effects are not additive (DA RhoA (0.4μM) + sJMD (1μM) diamonds 20.8 ± 2.5, n=10). E) Voltage dependence of activation obtained by plotting normalized tail current amplitude (I/Imax) against the voltage step. The continuous lines represent a Boltzmann fit of the data for control (circles), sJMD (open triangles), and DA RhoA (open squares). No difference in the activation profile was detected between treatments. Values are expressed as mean ± SE. F) Summary histogram showing averaged peak Ca²⁺ current density values for each condition tested in C and E expressed as a percentage of controls (100%). * Note significant decreases in HVA Ca²⁺ current influx for sJMD (p<0.001), DA RhoA (p<0.001), and DA RhoA + sJMD (p<0.001) associated conditions.