CIM muscle expresses the Na
1.5 α subunit and a Na
1.4 α subunit altered in its glycosylation. ( A) Western blots were performed on membranes prepared from gastrocnemius muscles of individual control or CIM rats using antibodies specific to the NaV 1.4 and 1.5 sodium channel isoforms or a pan-NaV 1.x antibody. Heart was a positive control for NaV 1.5. The mobility of the NaV 1.4 changed in CIM (see the upper arrow to normal channel versus lower arrowhead pointing to altered). It should be noted that 5% gels were used for the blots with the NaV 1.4 and 1.5 antibodies, while a 4% to 20% gel was used for the NaV 1.x antibody, thus reducing the apparent size difference between control and CIM for that antibody. (B) Quantification of blots with different sodium channel antibodies. The average expression of protein under control conditions was set as 100%, and expression in individual control and CIM was calculated relative to this average ( n = 4). Error bars shown are SEM, and asterisks indicate P < 0.01. Since the total amount of NaV 1.4 does not change in CIM, the increase observed with the pan-NaV 1.x is attributed to the up-regulation of the NaV 1.5. (C) Membranes from control and CIM muscles were treated with either PNGase F (to remove all N-linked glycosylation) or Neuraminidase (to remove only terminal sialic acid) and visualized by western blotting with a NaV 1.4-specific Ab on 5% gels. Treatment with PNGase F eliminated the migration difference between control and CIM channel, while Neuraminidase did not. (D) Neuraminidase treatment of fetuin, a control protein that is highly sialyated, shifts the molecular weight, indicating that the enzyme was functional.