Xiaowei Niu

Linker-Gating Ring Complex as Passive Spring and Ca2+-Dependent Machine for a Voltage- and Ca2+-Activated Potassium Channel

Ion channels are proteins that control the flux of ions across cell membranes by opening and closing (gating) their pores. It has been proposed that channels gated by internal agonists have an intracellular gating ring that extracts free energy from agonist binding to open the gates using linkers that directly connect the gating ring to the gates. Here we find for a voltage- and Ca(2+)-activated K+ (BK) channel that shortening the linkers increases channel activity and lengthening the linkers decreases channel activity, both in the presence and absence of intracellular Ca2+. These observations are consistent with a mechanical model in which the linker-gating ring complex forms a passive spring that applies force to the gates in the absence of Ca2+ to modulate the voltage-dependent gating. Adding Ca2+ then changes the force to further activate the channel. Both the passive and Ca(2+)-induced forces contribute to the gating of the channel.

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

Large-conductance Ca2+–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.

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca2

BK channels (Slo1) are widely distributed K+ channels that control Ca2+-dependent processes and cellular excitability. Their activation by intracellular Ca2+ (Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}) is highly cooperative, with Hill coefficients of typically 2–5. To investigate the cooperativity contributed by each of the four α subunits that form the BK channel, we studied single channels comprised of mixtures of functional subunits and subunits with a mutation to disrupt a key site (Ca-bowl) required for activation by low concentrations of Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}. As the number of functional subunits increased, we found a stepwise increase in the Hill coefficient of 0.3–0.8 per functional subunit and a stepwise decrease in the Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document} required for half activation (Kd). These results show directly that BK channels can open with 0, 1, 2, 3, or 4 functional Ca-bowls, and that each subunit with a functional Ca-bowl contributes a stepwise increase to both the cooperativity of activation and the apparent Ca2+ affinity. A model with 0–4 high-affinity allosteric activators and four low-affinity allosteric activators was examined. In this model, Ca2+ bindings were independent of one another and the cooperativity arose from the joint action of the allosteric activators on the open–closed equilibrium. Although this model described well the major features of the experimental data, some differences between the observed and predicted results indicated that additional factors not included in the model also contribute to the cooperativity.

Intra- and Intersubunit Cooperativity in Activation of BK Channels by Ca2+

The activation of BK channels by Ca2+ is highly cooperative, with small changes in intracellular Ca2+ concentration having large effects on open probability (Po). Here we examine the mechanism of cooperative activation of BK channels by Ca2+. Each of the four subunits of BK channels has a large intracellular COOH terminus with two different high-affinity Ca2+ sensors: an RCK1 sensor (D362/D367) located on the RCK1 (regulator of conductance of K+) domain and a Ca-bowl sensor located on or after the RCK2 domain. To determine interactions among these Ca2+ sensors, we examine channels with eight different configurations of functional high-affinity Ca2+ sensors on the four subunits. We find that the RCK1 sensor and Ca bowl contribute about equally to Ca2+ activation of the channel when there is only one high-affinity Ca2+ sensor per subunit. We also find that an RCK1 sensor and a Ca bowl on the same subunit are much more effective in increasing Po than when they are on different subunits, indicating positive intrasubunit cooperativity. If it is assumed that BK channels have a gating ring similar to MthK channels with alternating RCK1 and RCK2 domains and that the Ca2+ sensors act at the flexible (rather than fixed) interfaces between RCK domains, then a comparison of the distribution of Ca2+ sensors with the observed responses suggest that the interface between RCK1 and RCK2 domains on the same subunit is flexible. On this basis, intrasubunit cooperativity arises because two high-affinity Ca2+ sensors acting across a flexible interface are more effective in opening the channel than when acting at separate interfaces. An allosteric model incorporating intrasubunit cooperativity nested within intersubunit cooperativity could approximate the Po vs. Ca2+ response for eight possible subunit configurations of the high-affinity Ca2+ sensors as well as for three additional configurations from a previous study.

Slo1 Tail Domains, but Not the Ca2+ Bowl, Are Required for the β1 Subunit to Increase the Apparent Ca2+ Sensitivity of BK Channels

Functional large-conductance Ca2+- and voltage-activated K+ (BK) channels can be assembled from four α subunits (Slo1) alone, or together with four auxiliary β1 subunits to greatly increase the apparent Ca2+ sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the α subunit, which includes the RCK2 (regulator of K+ conductance) domain and Ca2+ bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca2+ bowl and high affinity Ca2+ sensitivity. In the second, the Ca2+ bowl was disrupted by mutations that greatly reduce the apparent Ca2+ sensitivity. We found that the β1 subunit increased the apparent Ca2+ sensitivity of Slo1 channels, independently of whether the α subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca2+ bowl was mutated. In contrast, β1 subunits no longer increased Ca2+ sensitivity when Slo1 tails were replaced by Slo3 tails. The β1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 β-estradiol activated these channels in the presence of β1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca2+ sensitivity induced by the β1 subunit does not require either the Ca2+ bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The β1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the β1 subunit–induced increase in apparent Ca2+ sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the β1 subunit may be different.

Ring of Negative Charge in BK Channels Facilitates Block by Intracellular Mg2+ and Polyamines through Electrostatics

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.

Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume.

Large-conductance Ca2+- and voltage-activated K+ (BK) channels have the largest conductance (250–300 pS) of all K+-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA− increased currents and MTSET+ decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K+]i. Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.