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Properties of Cloned Rat α1A Calcium Channels Transiently Expressed in the COS‐7 Cell Line
Author(s) -
Berrow N. S.,
Brice N. L.,
Tedder I.,
Page K. M.,
Dolphin A. C.
Publication year - 1997
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1111/j.1460-9568.1997.tb01422.x
Subject(s) - immunocytochemistry , calcium channel , transfection , calcium , voltage dependent calcium channel , microbiology and biotechnology , biophysics , chemistry , complementary dna , patch clamp , electrophysiology , biology , biochemistry , endocrinology , gene , neuroscience , organic chemistry
The rat brain α1A calcium channel clone has been expressed in COS‐7 cells together with the neuronal accessory subunits p1b and α2‐δ. 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 (α1, α2‐δ and β1b) 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 α1A/α/β1ba24IPlb 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 α1A current was completely blocked by 100 μM Cd 2+ and was also blocked by ω‐conotoxin MVIIC (500 nM). Dose‐inhibition curves and analysis of k on and k for for ω‐agatoxin IVA both revealed apparent K D values of approximately 11 nM for α1A currents, with a k D of 7.8 × 10 4 M ‐1 s ‐1 . The results suggest that α1A 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 ω‐agatoxin IVA 2–5 fold lower than reported for P current, but approximately 9‐fold higher than reported for Q current.