Ring of Negative Charge in BK Channels Facilitates Block by Intracellular Mg(2+) and Polyamines through Electrostatics

Intracellular Mg(2+) 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 Mg(2+) and polyamine concentrations. Removing the ring of charge greatly decreased the block, increasing K (B) (ap) (0 mV) for Mg(2+) 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 Mg(2+) and spermine block, and were unchanged by removing the ring of charge. When KCl(i) was increased from 150 mM to 3 M, the ring of charge no longer facilitated block, Mg(2+) 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 Mg(2+) 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 Mg(2+) or polyamines, while increasing the concentration of blocker available to enter and block the conduction pathway.