To test the hypothesis that intracellular Ca²⁺activation of large-conductance Ca²⁺-activated K⁺ (BK) channels involves the cytosolic form of phospholipase A₂(cPLA₂), we first inhibited the expression of cPLA₂ by treating GH₃ cells with antisense oligonucleotides directed at the two possible translation start sites on cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression of cPLA₂ in antisense-treated cells. We then examined the effects of intracellular Ca²⁺ concentration ([Ca²⁺]_i) on single BK channels from these cells. Open channel probability ( P _o) for the cells exposed to cPLA₂ antisense oligonucleotides in 0.1 μM intracellular Ca²⁺ was significantly lower than in untreated or sense oligonucleotide-treated cells, but the voltage sensitivity did not change (measured as the slope of the P _o-voltage relationship). In fact, a 1,000-fold increase in [Ca²⁺]_ifrom 0.1 to 100 μM did not significantly increase P _oin these cells, whereas BK channels from cells in the other treatment groups showed a normal P _o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acid on the P _oof BK channels from antisense-treated cells. Although arachidonic acid did significantly increase P _o, it did so without restoring the [Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels in GH₃ cells, the steep P _o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

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