The Study On Gating Mechanism, Trafficking And Binding Sites Of BK Channel

Large-conductance voltage- and Ca2+-activated K+ (BK) channels encoded by the msloαandβ2 subunits exist abundantly in rat chromaffin cells, pancreaticβcells and DRG neurons. The extracellular loop of hβ2 acting as the channel regulator influences the rectification and toxin sensitivity of BK channels and the inactivation domain at its N-terminus induces rapid inactivation. However, the regulatory mechanism, especially, the trafficking mechanism of hβ2 is still unknown. With the help of immunofluorescence and patch-clamp techniques, we found that the hβ2 subunit could be completely restrained within the cytoplast of cells by inserting three amino acids FIW at the upstream of its N-terminus termed double FIW or dFIW-hβ2. The dFIW-hβ2 also prevented mSloαsubunit from trafficking onto membrane surface and thus suppressed BK currents to null. Consequently, a novel method has been developed to identify the binding sites between the mSloαand hβ2 subunits of BK channels in this study. Our results reveal that the mSloαsubunits associate with the hβ2 subunits before they traffic to membrane surface; the residues E44D45 (hβ2) is a major binding site for mSloαand hβ2 subunits of BK channels and the residues D16E17 (hβ2) is a candidate as a preinactivation site. Our results also demonstrate that the weak binding sites ofαand hβ2 subunits of BK channels exist.We determine that the hβ2 subunit alone resides in the endoplasmic reticulum (ER), suggesting that trafficking mechanism of hβ2 differs from that of hβ1 opposite to what we predicted previously. We further demonstrate that a four-turnαhelical segment at the N terminus of hβ2 prevents the surface expression of hβ2, that is, the helical segment itself is a retention signal. Using the c-myc epitope tagged the extracellular loop of hβ2, we reveal that the most accessible site by antibody is located at the middle of the extracellular loop, which might provide clues to understand how the auxiliaryβsubunits regulates the toxin sensitivity and the rectification of BK-type channels.Single large-conductance calcium-activated K+ (BK) channels encoded by mSlo gene usually have a concerted gating, but a Drosophila--dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A)--exhibits very flickery openings. To probe this difference in the gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to that of dSlo1A. Rectification of the single channel current-voltage relationship of I323T decreased as [Ca2+]in increased from 10 to 300μM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating i.e. increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic, pore-lining residues at position 323 of mSlo act cooperatively in a"shutter-like"mechanism gating the permeation of K+ ions. We suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.