Large conductance Ca²⁺-activated K⁺ (BK) channels recorded from acutely dissociated rat CA1 hippocampal neurons exhibit a slow (41 ± 9 s in 10 µM Ca²⁺, mean ± S.E.M., n = 12) Ca²⁺-dependent inactivation (Hicks & Marrion, 1998). Inactivation was reversed by membrane hyperpolarization and when [Ca²⁺]_i was reduced to resting levels (100 nM), and permanently removed by the application of intracellular trypsin. These data suggest that inactivation of BK channels results from the intracellular block of the channel pore by an associated particle (Hicks & Marrion, 1998).

An inhibitor of Ca²⁺/calmodulin-dependent protein phosphatase 2B (PP2B), calcineurin, significantly delayed this slow inactivation behaviour. However, this appeared to be independent of protein dephosphorylation as application of ATP alone, or in combination with cAMP and the catalytic subunit of PKA did not alter the channel’s inactivation properties. Application of a pre-activated Ca²⁺/calmodulin-independent form of calcineurin (CaN₄₂₀) to inside-out patches excised from cultured hippocampal neurons mimicked the kinetic properties of BK channel inactivation. This effect was reversible in the absence of ATP, further underlying the dephosphorylation-independent nature of this activity. Calcineurin would appear to be regulating BK channel inactivation by a mechanism independent of protein dephosphorylation.

A direct association of the BK channel with calcineurin was therefore investigated in rat brain. A polyclonal antiserum raised against the C-terminal residues 1118-1135 of mSlo; a region conserved in rSlo (rat BK channel) immunoprecipitated a number of different proteins from solubilized rat brain. This antiserum immunoprecipitated the A subunit of calcineurin (58 kDa) in addition to the BK channel. Conversely, an antibody directed against the carboxyl terminus of the calcineurin Aα subunit immunoprecipitated the BK channel (125 kDa) from solubilized rat brain membranes. These data demonstrate a direct protein-protein interaction between the BK channel and calcineurin in rat brain, suggesting that constitutively bound calcineurin promotes BK channel inactivation by a mechanism independent of protein dephosphorylation. All animals were humanely killed.