Inhibition of a mammalian large conductance, calcium-sensitive K+ channel by calmodulin-binding peptides

1. The large conductance, calcium-sensitive K+ channel (BKCa channel) is a voltage-activated ion channel in which direct calcium binding shifts gating to more negative cellular membrane potentials. We hypothesized that the calcium-binding domain of BKCa channels may mimic the role played by calmodulin (CaM) in the activation of calcium-CaM-dependent enzymes, in which a tonic inhibitory constraint is removed on CaM binding. 2. To examine such a hypothesis, we used peptides from the autoregulatory domains of CaM kinase II (CK291-317) and cNOS (the constitutive nitric oxide synthase; cNOS725-747) as probes for the calcium-dependent activation of murine BKCa channels transiently expressed in HEK 293 cells. We found that these CaM-binding peptides produced potent, time-dependent inhibition of mammalian BKCa channel current following voltage-dependent activation. Inhibition was observed in both the presence and the absence of cytosolic free calcium. 3. Similar application of CK291-317 had no effect on either the amplitude or kinetics of voltage-dependent, macroscopic currents recorded from rabbit smooth muscle Kv1.5 potassium channels transiently expressed in HEK 293 cells. 4. Cytosolic application of both CK291-317 and tetraethylammonium (TEA) produced an additive and non-competitive block of BKCa current. This finding suggests that the peptide-binding site is distinct (e.g. outside the pore region of the channel) from that of TEA. 5. Our results are thus consistent with a model in which the BKCa channel's voltage-dependent gating process is under an intramolecular constraint that is relieved upon calcium binding. The intrinsic calcium sensor of the channel may thus interact with an inhibitory domain present in the BKCa channel, and by doing so, remove an inhibitory 'constraint' that permits voltage-dependent gating to occur at more negative potentials.