Abstract 3499: Extracellular Loop (Domain I, S5-S6) of α1D L-type Ca Channel is an Antigenic Site in Autoimmune Associated Congenital Heart Block
Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have established that anti-SSA/Ro -SSB/La autoantibodies inhibit L-type α1D Ca current, ICa-L and cross-react with the α1D Ca channel protein. This study aims at identifying the possible binding sites on α1D protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four α1D Ca channel protein domains I–IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera from 118 mothers with CHB children and 28 control healthy sera were used in this study. Seventeen of 118 (14.4%) maternal CHB sera reacted with the extracellular loop of Domain I S5-S6 region (E1). In contrast, 2 of 28 (7%) healthy control sera reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D. reading of the positive sera establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera demonstrated inhibition (44.1%) of the α1D ICa-L expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 extracellular loop fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel α1D subunit as a target for autoantibodies from mothers with CHB children. This novel finding provides insights into the development of therapeutic peptides that could bind to the pathogenic antibodies.