Properties of cloned rat alpha1A calcium channels transiently expressed in the COS-7 cell line

Eur J Neurosci. 1997 Apr;9(4):739-48. doi: 10.1111/j.1460-9568.1997.tb01422.x.


The rat brain alpha1A calcium channel clone has been expressed in COS-7 cells together with the neuronal accessory subunits beta1b and alpha2-delta. From reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and electrophysiology experiments, we have obtained no evidence that these cells contain any endogenous calcium channels. Transfected cells were identified by co-expression of a cDNA for the reporter Green Fluorescent Protein. From immunocytochemical evidence, a high degree of co-expression was obtained between Green Fluorescent Protein and individual calcium channel subunits. When all three calcium channel subunits (alpha1, alpha2-delta and beta1b) were co-expressed, evidence was obtained that all subunits were present at the cell membrane. Voltage-dependent calcium currents were observed between 24 and 72 h after transfection with the three calcium channel subunits. The current density for the combination alpha1A/alpha2-delta/beta1b was 4.19 +/- 0.69 pA.pF(-1) and the current produced was slowly inactivating. The time constant of inactivation of the maximum I(Ba) was 332 +/- 46 ms (n = 5). The voltage-dependence of activation and steady-state inactivation had voltages of half activation and inactivation of 9.5 +/- 2.5 mV and -30.4 +/- 1.5 mV respectively, and there was little overlap between the two curves. The alpha1A current was completely blocked by 100 microM Cd2+ and was also blocked by omega-conotoxin MVIIC (500 nM). Dose-inhibition curves and analysis of k(on) and k(off) for omega-agatoxin IVA both revealed apparent K(D) values of approximately 11 nM for alpha1A currents, with a k(on) of 7.8 x 10(4) M(-1).s(-1). The results suggest that alpha1A expressed in these cells has some resemblance to the P type component of calcium current observed in native neurons, although it shows a somewhat greater degree of inactivation than native P current, more similar to the Q type current component. It also has an affinity for omega-agatoxin IVA 2-5 fold lower than reported for P current, but approximately 9-fold higher than reported for Q current.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism
  • COS Cells
  • Cadmium / pharmacology
  • Calcium Channel Blockers / pharmacology
  • Calcium Channels / biosynthesis
  • Calcium Channels / physiology*
  • Cloning, Molecular
  • Genes, Reporter
  • Green Fluorescent Proteins
  • Luminescent Proteins / biosynthesis
  • Macromolecular Substances
  • Membrane Potentials
  • Molecular Sequence Data
  • Nerve Tissue Proteins / biosynthesis
  • Nerve Tissue Proteins / physiology*
  • Polymerase Chain Reaction
  • Protein Biosynthesis
  • Rats
  • Recombinant Fusion Proteins / biosynthesis
  • Spider Venoms / pharmacology
  • Transcription, Genetic
  • Transfection
  • omega-Agatoxin IVA


  • Cacna1a protein, rat
  • Calcium Channel Blockers
  • Calcium Channels
  • Luminescent Proteins
  • Macromolecular Substances
  • Nerve Tissue Proteins
  • Recombinant Fusion Proteins
  • Spider Venoms
  • omega-Agatoxin IVA
  • Cadmium
  • Green Fluorescent Proteins

Associated data

  • GENBANK/V01380