Functional Coupling of the β1 Subunit to the Large Conductance Ca2+-Activated K+ Channel in the Absence of Ca2+: Increased Ca2+ Sensitivity from a Ca2+-Independent Mechanism

Coexpression of the β1 subunit with the α subunit (mSlo) of BK channels increases the apparent Ca2+ sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca2+ sensitivity requires Ca2+, by comparing the gating in 0 Ca2+i of BK channels composed of α subunits to those composed of α+β1 subunits. The β1 subunit increased burst duration ∼20-fold and the duration of gaps between bursts ∼3-fold, giving an ∼10-fold increase in open probability (Po) in 0 Ca2+i. The effect of the β1 subunit on increasing burst duration was little changed over a wide range of Po achieved by varying either Ca2+i or depolarization. The effect of the β1 subunit on increasing the durations of the gaps between bursts in 0 Ca2+i was preserved over a range of voltage, but was switched off as Ca2+i was increased into the activation range. The Ca2+-independent, β1 subunit-induced increase in burst duration accounted for 80% of the leftward shift in the Po vs. Ca2+i curve that reflects the increased Ca2+ sensitivity induced by the β1 subunit. The Ca2+-dependent effect of the β1 subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the β1 subunit are independent of Ca2+i suggests that the β1 subunit mainly alters the energy barriers of Ca2+-independent transitions. The changes in gating induced by the β1 subunit differ from those induced by depolarization, as increasing Po by depolarization or by the β1 subunit gave different gating kinetics. The complex gating kinetics for both α and α+β1 channels in 0 Ca2+i arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca2+i

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

Ball-and-chain inactivation in a calcium-gated potassium channel

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a ‘ball-and-chain’ mechanism3–7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum—a purely calcium-gated and inactivating channel—in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism

Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites

Membrane phospholipids control gating of the mechanosensitive potassium leak channel TREK1

Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators

An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a “microbody,” in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of β coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses