Ca channels induced in Xenopus oocytes by rat brain mRNA

Year: 1987

DOI: 10.1523/jneurosci.07-03-00875.1987

PMCID: PMC6569056

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2–5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline.

I_(Ba) in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 ± 4 nA, n = 21). In contrast, injected oocytes showed a peak I_(Ba) of 339 ± 42 nA (n = 33). The threshold for activation of I_(Ba) was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (τ = 650 ± 50 msec at +20 mV). The maintained current was 67 ± 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward I_(Ba) could be observed in BaCl₂ solutions at potentials positive to E_(Cl) and also in Na-free salines, indicating that neither Cl⁻ nor Na⁺ was carrying the inward current.

Although I_(Ba) displayed voltage- independent blockade by Cd (50% inhibition at 6 µM), the peptide Ca channel antagonist, ω-CgTX (1 µM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 µM, or when cells were held at -40 mV) or agonist Bay K-8644. However, I_(Ba) was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT₂). In contrast, use of forskolin to activate protein kinase A did not alter I_(Ba). However, experiments in the presence of Cd revealed that forskolin decreased I_K. Ca channels produced by rat brain mRNA were thus in contrast to the nifedipine-sensitive, Bay K-8644- and forskolin-enhanced Ca channels observed after injection of rat heart mRNA (Dascal et al., 1986).