Re-evaluation of the P/Q Ca2+ channel components of Ba2+ currents in bovine chromaffin cells superfused with solutions containing low and high Ba2+ concentrations

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Abstract
This study was undertaken to reassess the set of voltage-dependent Ca2+ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar concentrations of ω-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba2+. Whole-cell Ba2+ currents (IBa) through Ca2+ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of −80 mV and depolarising pulses to 0 mV. Mean peak IBa was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, ω-conotoxin MVIIC (3 μM) inhibited IBa by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of ω-agatoxin IVA (20 nM) inhibited IBa by 9%; 2 μM inhibited IBa by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 μM) and ω-conotoxin GVIA (1 μM), 2 μM ω-agatoxin IVA inhibited the remaining current (about 40–45%); this blockade was independent of the Ba2+ concentration. The current could be fully blocked by the cocktail furnidipine/gw-conotoxin GVIA/high ω-agatoxin IVA, both in low and high Ba2+ concentrations. The large Q-type channel component of IBa is blocked by micromolar concentrations of ω-agatoxin IVA and ω-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by ω-conotoxin MVIIC, the blockade by high concentrations of ω-agatoxin IVA was equally effective in solutions with a low or a high Ba2+ concentration. Hence, the use of appropriate Ba2+ and toxin concentrations in this study reveals that P-type Ca2+ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type Ca2+ channels. An R-type residual current is not present in these cells.