Figure 5

Na _V 1.4 co-precipitates with members of the dystrophin associated protein complex (DAPC). Using an antibody generated against a peptide corresponding to the highly conserved III-IV linker region (pan Na_V 1.x), sodium channels from NP40-solubilized control and CIM skeletal muscle membranes were immunoprecipitated. The immunoprecipitated (IP) control (Con) or CIM materials were resolved on SDS-PAGE gels and probed in western blots with the indicated antibodies. As a negative control, the IP was performed in the presence of blocking peptide (IP/pep), the same sequence used as the antigen; and as a positive control, starting membranes were used (Memb). (A) A full-panel western with the Na_V 1.4-specific monoclonal antibody LD3 is shown on the left in comparison to a protein gel stained with SYPRO Ruby protein stain on the right. The denatured antibody heavy chain (Ab HC) and light chain (Ab LC) are present in the immunoprecipitated samples, as best seen in the SYPRO stained gel. A large number of proteins co-precipitate with the sodium channel (Na_V 1.x), and many of these are shown to be specific since they are absent in the peptide control. (B) Using antibodies against plectin, dystrophin (dys), neuronal nitric oxide synthase (nNOS), syntrophin (syn), and β-dystroglycan (β-dys) confirms that many components of the DAPC are present in the control and CIM IPs. ( C) Quantification of the data from panel B. For each antibody in each CoIP, the signal for the control was set as 100% and the signal for the CIM was determined relative to this. The average of the signals for the CIM: control for each antibody is shown, and error bars are SEM ( n =6). For some antibodies, notably the sodium channel antibodies, approximately equal signals were seen in control and CIM CoIPs. For other antibodies, notably dystrophin and syntrophin, there were slightly elevated amounts of these proteins present in the CIM. Finally, for the nNOS and β-dystroglycan, there was considerably more protein in the CIM CoIPs. These results are summarized in cartoon form in ( D) which shows ‘tightly’ associated proteins in grey, and ‘loosely’ associated proteins in white. The protein associations shown in the cartoon are based on work carried out by a number of investigators, which previously demonstrated that Na_V 1.x channels associate with proteins of the DAPC through their consensus S/TXV-COOH C-termini [26, 27]. The dynamic regulation of the signaling protein nNOS in CIM suggests that it may play a role in the disease process, including affecting the inactivation gating of the adjacent sodium channels.