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Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, FL 33101

Correspondence to Karl L. Magleby: kmagleby{at}miami.edu

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 KCl_i 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.

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