VOLTAGE-DEPENDENT BINDING AND CALCIUM-CHANNEL CURRENT INHIBITION BY AN ANTI-ALPHA(1D) SUBUNIT ANTIBODY IN RAT DORSAL-ROOT GANGLION NEURONS AND GUINEA-PIG MYOCYTES

1. The presence of calcium channel alpha(1D) subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea-pig cardiac myocyte s was demonstrated using the reverse transcriptase-polymerase chain re action. 2. An antipeptide antibody targeted at a region of the voltage -dependent calcium channel alpha(1D) subunit C-terminal to the pore-fo rming SS1-SS2 loop in domain IV (amino acids 1417-1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were d epolarized with 30 mM K+. 3. Incubation of cells under depolarizing co nditions for 2-4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.0 5) for cardiac myocytes when compared with controls. 4. S-(-)-Bay K 86 44 (1 mu M) enhanced calcium channel currents in DRGs by 75 +/- 19% (n = 5) in neurones incubated under depolarizing conditions with antibod y that had been preadsorbed with its immunizing peptide (100 mu g ml(- 1)). This was significantly (P < 0.05) larger than the enhancement by S-(-)-Bay K 8644 that was seen with cells incubated under identical co nditions but with antibody alone, which was 15 +/- 4% (n = 5). 5. Thes e results demonstrate the presence of calcium channel alpha(1D) subuni ts in rat DRG neurones and guinea-pig cardiac myocytes. They also show that amino acids 1417-1434 of the alpha(1D) subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calci um channel current and reduces the ability of S-(-)-Bay K 8644 to enha nce this current, indicating that it is an L-type current that is atte nuated.