Abstract
BK channels are regulated by two distinct physiological signals, transmembrane potential and intracellular Ca(2+), each acting through independent modular sensor domains. However, despite a presumably central role in the coupling of sensor activation to channel gating, the pore-lining S6 transmembrane segment has not been systematically studied. Here, cysteine substitution and modification studies of the BK S6 point to substantial differences between BK and Kv channels in the structure and function of the S6-lined inner pore. Gating shifts caused by introduction of cysteines define a pattern and direction of free energy changes in BK S6 distinct from Shaker. Modification of BK S6 residues identifies pore-facing residues that occur at different linear positions along aligned BK and Kv S6 segments. Periodicity analysis suggests that one factor contributing to these differences may be a disruption of the BK S6 α-helix from the unique diglycine motif at the position of the Kv hinge glycine. State-dependent MTS accessibility reveals that, even in closed states, modification can occur. Furthermore, the inner pore of BK channels is much larger than that of K(+) channels with solved crystal structures. The results suggest caution in the use of Kv channel structures as templates for BK homology models, at least in the pore-gate domain.