Voltage-dependent binding and calcium channel current inhibition by an anti-alpha 1D subunit antibody in rat dorsal root ganglion neurones 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 myocytes was demonstrated using the reverse transcriptase-polymerase chain reaction. 2. An antipeptide antibody targeted at a region of the voltage-dependent calcium channel alpha 1D subunit C-terminal to the pore-forming SS1-SS2 loop in domain IV (amino acids 1417-1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+. 3. Incubation of cells under depolarizing conditions 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 < 0.05) for cardiac myocytes when compared with controls. 4. S-(-)-Bay K 8644 (1 microM) enhanced calcium channel currents in DRGs by 75 +/- 19% (n = 5) in neurones incubated under depolarizing conditions with antibody that had been preabsorbed with its immunizing peptide (100 micrograms 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 conditions but with antibody alone, which was 15 +/- 4% (n = 5). 5. These results demonstrate the presence of calcium channel alpha 1D subunits 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 calcium channel current and reduces the ability of S-(-)-Bay K 8644 to enhance this current, indicating that it is an L-type current that is attenuated.
Wyatt
C N
CN
Department of Pharmacology, Royal Free Hospital School of Medicine, London, UK.
Campbell
V
V
Brodbeck
J
J
Brice
N L
NL
Page
K M
KM
Berrow
N S
NS
Brickley
K
K
Terracciano
C M
CM
Naqvi
R U
RU
MacLeod
K T
KT
Dolphin
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D002478:Cells, Cultured; D017931:DNA Primers; D004594:Electrophysiology; D000939:Epitopes; D005727:Ganglia, Spinal / Q000502:physiology*; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D006352:Heart Ventricles; D007150:Immunohistochemistry; D008297:Male; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D015220:Calcium Channels; D017931:DNA Primers; D000939:Epitopes