Ring of Negative Charge in BK Channels Facilitates Block by Intracellular Mg2+ and Polyamines through Electrostatics

Abstract

Intracellular Mg2+ and natural polyamines block outward currents in BK channels in a highly voltage-dependent manner. Here we investigate the contribution of the ring of eight negatively charged residues (4 x E321/E324) at the entrance to the inner vestibule of BK channels to this block. Channels with or without (E321N/E324N) the ring of negative charge were expressed in oocytes and unitary currents were recorded from inside-out patches over a range of intracellular Mg2+ and polyamine concentrations. Removing the ring of charge greatly decreased the block, increasing K B ap (0 mV) for Mg2+ block from 48.3 ± 3.0 to 143 ± 8 mM, and for spermine block from 8.0 ± 1.0 to 721 ± 9 mM (150 mM symmetrical KCl). Polyamines with fewer amine groups blocked less: putrescine < spermidine < spermine. An equation that combined an empirical Hill function for block together with a Boltzmann function for the voltage dependence of K B ap described the voltage and concentration dependence of the block for channels with and without the ring of charge. The Hill coefficients for these descriptions were <1 for both Mg2+ and spermine block, and were unchanged by removing the ring of charge. When KCli was increased from 150 mM to 3 M, the ring of charge no longer facilitated block, Mg2+ block was reduced, spermine block became negligible, and the Hill coefficients became ∼1.0. BK channels in cell-attached oocyte patches displayed inward rectification, which was reduced for channels without the ring of charge. Taken together, these observations suggest that the ring of negative charge facilitates block through a preferential electrostatic attraction of Mg2+ and polyamine over K+. This preferential attraction of multivalent blockers over monovalent K+ would decrease the K+ available at the inner vestibule to carry outward current in the presence of Mg2+ or polyamines, while increasing the concentration of blocker available to enter and block the conduction pathway.