title: 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 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 approximately 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.
authors.affiliation: Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, FL 33101, USA.
authors.forename: Yaxia
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Niu
authors.forename: Tinatin I
authors.initials: TI
authors.lastname: Brelidze
authors.forename: Karl L
authors.initials: KL
authors.lastname: Magleby
mesh_terms: D000465:Algorithms; D000818:Animals; D005260:Female; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000502:physiology; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D011073:Polyamines / Q000378:metabolism* / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000502:physiology; D011700:Putrescine / Q000494:pharmacology; D018075:RNA, Complementary / Q000235:genetics; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D055672:Static Electricity; D014982:Xenopus laevis
chemical_list: C465449:Kcnma1 protein, mouse; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D011073:Polyamines; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary; D013096:Spermine; D008274:Magnesium; D011188:Potassium; D013095:Spermidine; D011700:Putrescine