Single Aspartate Substitutions at Multiple Deep-Pore Positions Lead to Constitutively Open BK Channels

Abstract

The pore-lining amino acids of ion channel proteins reside on the interface between a polar (the pore) and a non-polar environment (the rest of the protein). Manipulating the charge states of their side-chains, by tilting the energy balance between a buried and a pore-exposed conformation, can reveal important details about gating-related structural dynamics. For example, charged side-chains at the M314 position in the BK channel stabilize an open conformation. The M314D mutant, in particular, led to constitutive channel activities. We have now scanned the S6 region (I308 to N328) with aspartate replacement. We found 3 other positions where aspartate substitutions led to constitutively open BK channels in nominal zero calcium and neutral pH. These mutants are L312D, A313D and A316D. Together with M314, these positions are all located within the deep-pore region of the channel, which is immediately intracellular to the selectivity filter but more extracellular to the lower-pore region that aligns with the Kv channel “bundling crossing”. These findings provide further evidence that the BK channel pore is different from that of Kv channels, where aspartate substitution at a single position (as opposed to multiple positions in BK) in the lower-pore region (as opposed to the deep-pore region in BK) led to constitutive channel activities. The aspartate mediated constitutive activities in Kv channels is believed to result from the disruption of specific hydrophobic interactions in the “bundle-crossing” gate of a closed channel. Our observations with BK channels are unlikely to result from disruption of specific interactions, as multiple positions in the BK deep-pore, some of them adjacent along the S6 helix, showed aspartate mediated constitutive activities.