Properties of cloned rat alpha1A calcium channels transiently expressed in the COS-7 cell line.
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.
Berrow
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Department of Pharmacology, Royal Free Hospital School of Medicine, London, UK.
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D000818:Animals; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002104:Cadmium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000096:biosynthesis / Q000502:physiology*; D003001:Cloning, Molecular; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis; D046911:Macromolecular Substances; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000502:physiology*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013111:Spider Venoms / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D020867:omega-Agatoxin IVA; D020864:Calcium Channels, N-Type
D002104:Cadmium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D013111:Spider Venoms; D020867:omega-Agatoxin IVA; C491367:Cacna1a protein, rat; D020864:Calcium Channels, N-Type