Exploring Whether a Large Entrance To The Inner Vestibule Of BK Channels Is Required For Their Large Conductance

Abstract

To explore whether a large entrance to the inner vestibule of BK channels is required for their large conductance, we examine if changing the size of the entrance alters the single-channel outward current, gamma. Previous studies suggest that E321/E324 in BK channels are located at the entrance to the inner vestibule. To test if E321/E324 are accessible to intracellular ions, we compare gamma of E321C and E324C before and after treatment with maleimido-proprionic-acid (MPA), a membrane impermeable negatively charged thiol reagent. Gamma for E321C-MPA and E324C-MPA increases by ∼20%, presumably due to the added negative charge on MPA attracting K+ ions to the inner vestibule. This suggests that E321/E324 are accessible to the conduction pathway. At the same time, gamma for E321C-MPA and E324C-MPA is still ∼10% less than for wt channels, suggesting that the larger size of C-MPA compared to glutamic acid restricts current flow, although we cannot exclude that the location of the negative charge on MPA vs. glutamic acid attracts fewer K+ ions to the inner vestibule. We also change the size of the entrance to the inner vestibule by substituting residues with different sized side chains at E321/E324. For hydrophobic substitutions, tryptophan with the largest volume has a ∼30% smaller gamma than substitutions with alanine, valine, and leucine. For hydrophilic substitutions (serine, threonine, asparagine, and glutamine), larger side chains appear to decrease gamma, but any effects are small. Increasing [K+]i from 0.15 to 2.5 M removes all differences in gamma associated with different sized side chains. All substitutions have little effect on inward current. These observations suggest that a large entrance to the inner vestibule of BK channels contributes to their large conductance.