A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification

Abstract

Large-conductance Ca 2؉ -voltage-activated K ؉ channels (BK channels) control many key physiological processes, such as neurotransmitter release and muscle contraction. A signature feature of BK channels is that they have the largest single channel conductance of all K ؉ channels. Here we examine the mechanism of this large conductance. Comparison of the sequence of BK channels to lower-conductance K ؉ channels and to a crystallized bacterial K ؉ channel (MthK) revealed that BK channels have a ring of eight negatively charged glutamate residues at the entrance to the intracellular vestibule. This ring of charge, which is absent in lower-conductance K ؉ channels, is shown to double the conductance of BK channels for outward currents by increasing the concentration of K ؉ in the vestibule through an electrostatic mechanism. Removing the ring of charge converts BK channels to inwardly rectifying channels. Thus, a simple electrostatic mechanism contributes to the large conductance of BK channels.L arge-conductance Ca 2ϩ -voltage-activated K ϩ (BK) channels are activated in a highly synergistic manner by intracellular Ca 2ϩ (Ca i 2ϩ ) and membrane depolarization (1)(2)(3)(4)(5)(6)(7)(8)(9). When open, the efflux of K ϩ out of the cell hyperpolarizes the membrane potential, turning off voltage-dependent Ca 2ϩ channels and reducing the influx of Ca 2ϩ available to both activate BK channels and control cellular processes. This negative feedback mechanism allows BK channels to play a key role in regulating many physiological processes, such as neurotransmitter release (10, 11), repetitive firing of neurons (12), spike broadening during repetitive firing (13), the electrical tuning of hair cells in the cochlea (14,15), and muscle contraction (16).BK channels (for big K ϩ ) have the largest single-channel conductance of all K ϩ selective channels, being 250-300 pS in symmetrical 150 mM KCl (8,[17][18][19][20]. Like most K ϩ channels of lower conductance, BK channels have a tetrameric structure, with four ␣ subunits forming functional channels. BK channels also have the same selectivity filter sequence (GYG) found in most other K ϩ channels of lower conductance (21). Thus, the question arises as to why the conductance of BK channels is so big.Previous experimental and theoretical work has suggested that charged residues located in the vestibules and pores of ion channels can play a major role in controlling the unitary conductance through an electrostatic mechanism (22-30). Such rings of fixed charge could increase the concentration of the permeating ions in the vestibules of the channels, leading to increased availability of ions to transit the selectivity filter, which would increase the unitary (single-channel) conductance.In this study we show by comparison of the sequence of BK channels to lower-conductance K ϩ channels and to a bacterial K ϩ channel (MthK) crystallized in the open state (31) that BK channels have a ring of eight negatively charged glutamate residues at the entrance to the intracellular vestibule that ...

Crucial Points within the Pore as Determinants of K+ Channel Conductance and Gating

“…4A); the higher reduction of outward current renders the channel inward rectified in the absence of Ca 2+ (Fig. 4C), similar to what was observed in BK channels 9 . Furthermore, the E92Q mutation also abolishes the Ca 2+ blockage on the intracellular side as shown by the plot of Ca 2+ dependent blockage of the outward current (Fig.…”

Inositol Trisphosphate Receptor Ca2+Release Channels

“…BK channels have one or two conserved acidic residues near the end of the pore inner helix that is absent in lower conductance K ϩ channels. Neutralization of these residues greatly reduced single-channel conductance, whereas introduction of the acidic residues into KcsA enhanced channel conductance into a range characteristic of BK channels (58,346). The magnitude of the conductance was correlated with the amount of negative charge in the inner vestibule.…”

The carboxyl-terminal region of cyclic nucleotide-modulated channels is a gating ring, not a permeation path

“…D487 and D489, however, face the central axis and are directly accessible to the water-filled center of the tunnel. Negative charge rings are a common feature at the mouths of cation channels, where they help to focus the permeant cations and likely act as a ''prefilter'' by repelling intracellular anions from the immediate vicinity of the pore (23)(24)(25)(26)(27)(28)(29)(30). Despite this architecture, it is possible that permeant ions leave the channel before reaching this tunnel (Fig.…”