Novel [alpha]-KTx Sites in the BK Channel and Comparative Sequence Analysis Reveal Distinguishing Features of the BK and KV Channel Outer Pore

The α-KTx peptide toxins inhibit different types of potassium channels by occluding the outer channel pore composed of four identical α subunits. The large-conductance, calcium-activated (BK or Slo1) and voltage-dependent (KV) potassium channels differ in their specificity for the different α-KTx subfamilies. While many different α-KTx subfamilies of different sizes inhibit KV1 channels with high affinity, only one subfamily, α-KTx 1.x, inhibits BK channels with high affinity. Two solvent-exposed regions of the outer pore that influence α-KTx binding, the turret and loop, display high sequence variability among different potassium channels and may contribute to differences in α-KTx specificity. While these α-KTx domains have been studied in KV1 channels, little is known about the corresponding BK α-KTx domains. To define α-KTx sites in the BK outer pore, we examined the effect of 19 outer pore mutations on specific binding of 125I-labeled iberiotoxion (IbTX or α-KTx 1.3) and on their cell-surface expression. Similar to α-KTx sites in the Shaker KV1 loop, site-directed mutations in the BK loop disrupted specific IbTX binding. In contrast, mutations in the BK turret region revealed three novel α-KTx sites, Q267, N268, and L272, which are distinct from α-KTx sites in the KV1 turret. The BK turret region shows no sequence identity with KV1 and MthK turrets of known 3D structure. To define the BK turret, we used secondary structure prediction methods that incorporated information from sequence alignment of 30 different Slo1 and Slo3 turret sequences from 5 of the 7 major animal phyla representing 27 different species. Results of this analysis suggest that the BK turret contains 18 amino acids and is defined by a cluster of strictly conserved polar residues at the N-terminal side of the turret. Thus, the BK turret is predicted to have six more amino acids than the KV1 turret. Results of this work suggest that BK and KV1 outer pores have a similar α-KTx domain in the loop preceding the inner helix, but that the BK turret comprises a unique α-KTx interaction surface that likely contributes to the exclusive selectivity of BK channels for α-KTx1.x toxins.