Antisense depletion of beta-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels.
1. The role of the voltage-dependent calcium channel (VDCC) beta-subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta-subunits in a number of preparations. Immunoreactivity for the VDCC beta-subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm. 2. A twenty-six-mer antisense oligonucleotide with homology to all published VDCC beta-subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta-subunit immunoreactivity was observed after 108 h suggesting a half-life for the turnover of the beta-subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide. 3. The effect of depletion of VDCC beta-subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co-expression of cloned beta- with alpha 1-subunits in oocytes and other expression systems. 4. The ability of the 1,4-dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta-subunit immunoreactivity. This result is in agreement with the finding in several systems that co-expression of the beta-subunit with alpha 1-subunits results in an increased number of DHP binding sites. 5. These results show that calcium channel beta-subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties.
Berrow
N S
NS
Department of Pharmacology, Royal Free Hospital School of Medicine, London, UK.
Campbell
V
V
Fitzgerald
E M
EM
Brickley
K
K
Dolphin
A C
AC
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004594:Electrophysiology; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D015640:Ion Channel Gating / Q000502:physiology; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D015220:Calcium Channels; D016376:Oligonucleotides, Antisense; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester