Fig. 2.
Interaction site between sodium channel α-subunits is located between amino acids 493–517. a Schematic representation of the different truncated GFP-Nav1.5. Constructs were generated from GFP-Nav1.5, where GFP is tagged to the N-terminus. Truncations were generated by inserting a stop codon at either amino acid 555, 517, 493, 470 or 450. b Current Density at −20 mV recorded from iCells transfected with either GFP or truncated GFP-fused Nav1.5 constructs as illustrated in a. Data are presented as mean ± SEM, ***p < 0.001. The name of the construct studied is indicated below each bar of the graph. c Co-immunoprecipitation were performed between the full-length Nav1.5 containing an HA tag and the same fragments illustrated in panel A and studied in panel B. HEK293 cells were co-transfected with HA-Nav1.5 and each of the fragments separately. Two first western blots show the total proteins isolated from the transfected HEK293 cells. GFP antibody probes for the truncated sodium channel constructs and the HA antibody probes for the full-length HA-Nav1.5. Co-immunoprecipitations were performed using the HA antibody and then revealed for GFP (truncated channel). The name of the truncated channel studied is illustrated above the first western. Importantly, the loss of interaction between α-subunits concurs with the loss of DN effect. Western is a representative example of four different experiments. d Co-immunoprecipation between truncated sodium channels illustrated in panel A and 14-3-3 were performed on total cell lysate (left) or plasma membrane preparation (right). Co-immunoprecipitations were performed using the 14-3-3 antibody and immunoprecipitations were revealed using GFP antibody to probe for interaction with the truncated sodium channels. The constructs studied in each lane are labeled at the top of panel c. In panel d, the top two westerns illustrate the total protein lysate and the last western shows the results of the co-immunoprecipitation. 14-3-3 interacts with the GFP-1-555X construct but not with the others. Westerns are representative examples of 5 different experiments. Full blots for C and D are presented in Supplementary Fig. 12. e Schematic representation of the region of interaction between α-subunits and between α-subunits and 14-3-3 protein in the DI-DII linker as supported from our data