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.

Structure and electrostatic property of cytoplasmic domain of ZntB transporter

“…Indeed as it was shown for other metal channels, electrostatic forces may play an important role in metal transport. [22][23][24] The cation selectivity of CorA and ZntB was attributed to the difference in their signature motifs (CorA:YGMNFxxMPEL, ZntB: GxxG[I,V]NxGGxP). 12,25 The motif is located between two transmembrane ahelices on the outer surface of the membrane and it tends to be disordered in the crystal structures.…”

Properties of Slo1 K + channels with and without the gating ring

“…Standard molecular biology methods (12), Xenopus oocyte expression systems (12,13,47), and electrophysiological techniques (12,13,21,48) were used. The indicated concentrations of Ca 2+ and Mg 2+ were applied to the intracellular membrane surface.…”

BK Channels – Focus on Polyamines, Ethanol/Acetaldehyde and Hydrogen Sulfide (H2S)

“…The ideas why the conductance of these channels may be so large despite their high selectivity for K + can be summarized as followed: a) a negatively charged ring structure at the inner face of the channel which by electrostatic attraction of K + to the entrance approximately doubles the current amplitude (Brelidze et al 2003;Nimigean et al 2003;Zhang et al, 2006;Carvacho et al, 2008), b) a voluminous inner cavity with an excess of negatively charged amino acids near the selectivity filter which traps K + and facilitates their entrance into the selectivity filter (Brelidze & Magleby 2005;Li & Aldrich 2004), and c) a ring of four negative charges at the extracellular mouth of the channel (Haug et al, 2004), which pulls K + from the channel. The exact mechanism by which the high conductance of these channels is accomplished is still not fully understood in particular the contribution of the later two mechanisms to channel conductance have to be tested rigorously.…”

“…A more detailed molecular explanation of how polyamines block BK channels was presented by Zhang et al, 2006. They found the ring of 8 negative charges at the inner channel mouth to be responsible for the attraction of polyamines to the channel pore.…”