Inhibitory effects of purified antibody against α-1 repeat(117-137) on Na~+-Ca~(2+) exchange and L-type Ca~(2+) currents in rat cardiomyocytes

100176, China Abstract: Considering that -1 repeat region may be involved in the ion binding and translocation of Na + -Ca 2+ exchanger (NCX), it is possible that the antibodies against NCX -1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against -1 repeat (117-137), designated as -1(117-137), on NCX activity. The antibody against the synthesized -1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na + - Ca 2+ exchange current (INa/Ca) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca 2+ current (ICa,L), voltage-gated Na + current (INa) and delayed rectifier K + current (IK) were also observed. The amino acid sequences of -1(117-137) in NCX and residues 1 076-1 096 within L-type Ca 2+ channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward INa/Ca were decreased by the antibody against -1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC50 values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased ICa,L in a concentration-dependent manner with IC50 of 22.7 nmol/L. No obvious effects of the antibody on INa and IK were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX -1(117-137) and residues 1 076-1 096 within L-type Ca 2+ channel. These results suggest that antibody against -1(117-137) is a blocking antibody to NCX and can also decrease ICa,L in a concentration-dependent manner, while it does not have obvious effects on INa and IK.