Let It Go and Open Up, an Ensemble of Ion Channel Active States.

Ligand binding usually moves the target protein from an ensemble of inactive states to a well-defined active conformation. Matthies et al. flip this scheme around, finding that, for the magnesium channel CorA, loss of ligand binding induces an ensemble of conformations that turn the channel on.

Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

EMC chaperone-CaV structure reveals an ion channel assembly intermediate.

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (CaVs)3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaVα1 (ref. 3), and the auxiliary CaVß5 and CaVα2δ subunits6,7. Here we present cryo-electron microscopy structures of human brain and cardiac CaV1.2 bound with CaVß3 to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)8,9-and of the assembled CaV1.2-CaVß3-CaVα2δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the CaVα2δ-interaction site. The structures identify the CaVα2δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs6, show that EMC and CaVα2δ interactions with the channel are mutually exclusive, and indicate that EMC-to-CaVα2δ hand-off involves a divalent ion-dependent step and CaV1.2 element ordering. Disruption of the EMC-CaV complex compromises CaV function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a CaV assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.

Definition of a saxitoxin (STX) binding code enables discovery and characterization of the anuran saxiphilin family.

American bullfrog (Rana castesbeiana) saxiphilin (RcSxph) is a high-affinity "toxin sponge" protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (NaVs). How specific RcSxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX "recognition code" enabled engineering of RcSxph to improve its ability to rescue NaVs from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by ∼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.

K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site.

Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit. In contrast to other potassium channels, K2P channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K2P selectivity filter gating.

Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

The Polysite Pharmacology of TREK K2P Channels.

K2P (KCNK) potassium channels form "background" or "leak" currents that have critical roles in cell excitability control in the brain, cardiovascular system, and somatosensory neurons. Similar to many ion channel families, studies of K2Ps have been limited by poor pharmacology. Of six K2P subfamilies, the thermo- and mechanosensitive TREK subfamily comprising K2P2.1 (TREK-1), K2P4.1 (TRAAK), and K2P10.1 (TREK-2) are the first to have structures determined for each subfamily member. These structural studies have revealed key architectural features that underlie K2P function and have uncovered sites residing at every level of the channel structure with respect to the membrane where small molecules or lipids can control channel function. This polysite pharmacology within a relatively small (~70 kDa) ion channel comprises four structurally defined modulator binding sites that occur above (Keystone inhibitor site), at the level of (K2P modulator pocket), and below (Fenestration and Modulatory lipid sites) the C-type selectivity filter gate that is at the heart of K2P function. Uncovering this rich structural landscape provides the framework for understanding and developing subtype-selective modulators to probe K2P function that may provide leads for drugs for anesthesia, pain, arrhythmia, ischemia, and migraine.

Global versus local mechanisms of temperature sensing in ion channels.

Ion channels turn diverse types of inputs, ranging from neurotransmitters to physical forces, into electrical signals. Channel responses to ligands generally rely on binding to discrete sensor domains that are coupled to the portion of the channel responsible for ion permeation. By contrast, sensing physical cues such as voltage, pressure, and temperature arises from more varied mechanisms. Voltage is commonly sensed by a local, domain-based strategy, whereas the predominant paradigm for pressure sensing employs a global response in channel structure to membrane tension changes. Temperature sensing has been the most challenging response to understand and whether discrete sensor domains exist for pressure and temperature has been the subject of much investigation and debate. Recent exciting advances have uncovered discrete sensor modules for pressure and temperature in force-sensitive and thermal-sensitive ion channels, respectively. In particular, characterization of bacterial voltage-gated sodium channel (BacNaV) thermal responses has identified a coiled-coil thermosensor that controls channel function through a temperature-dependent unfolding event. This coiled-coil thermosensor blueprint recurs in other temperature sensitive ion channels and thermosensitive proteins. Together with the identification of ion channel pressure sensing domains, these examples demonstrate that "local" domain-based solutions for sensing force and temperature exist and highlight the diversity of both global and local strategies that channels use to sense physical inputs. The modular nature of these newly discovered physical signal sensors provides opportunities to engineer novel pressure-sensitive and thermosensitive proteins and raises new questions about how such modular sensors may have evolved and empowered ion channel pores with new sensibilities.

Metabolic and thermal stimuli control K(2P)2.1 (TREK-1) through modular sensory and gating domains.

K(2P)2.1 (TREK-1) is a polymodal two-pore domain leak potassium channel that responds to external pH, GPCR-mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K(2P)2.1 (TREK-1) intracellular C-terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C-type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore-forming core, we further demonstrate that Ct is the primary heat-sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K(2P)2.1 (TREK-1) in which there is a clear crosstalk between the C-type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature-sensing domains in temperature-sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature-sensitive channels.

Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC).

Transmembrane proteins with unknown function 16 (TMEM16A) is a calcium-activated chloride channel (CaCC) important for neuronal, exocrine, and smooth muscle functions. TMEM16A belongs to a family of integral membrane proteins that includes another CaCC, TMEM16B, responsible for controlling action potential waveform and synaptic efficacy, and a small-conductance calcium-activated nonselective cation channel, TMEM16F, linked to Scott syndrome. We find that these channels in the TMEM16 family share a homodimeric architecture facilitated by their cytoplasmic N termini. This dimerization domain is important for channel assembly in eukaryotic cells, and the in vitro association of peptides containing the dimerization domain is consistent with a homotypic protein-protein interaction. Amino acid substitutions in the dimerization domain affect functional TMEM16A-CaCC channel expression, as expected from its critical role in channel subunit assembly.

Multiple modalities converge on a common gate to control K2P channel function.

Members of the K(2P) potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K(2P)s are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K(2P), K(2P)2.1 (KCNK2/TREK-1), at a common molecular gate that comprises elements of the pore-forming segments and the N-terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K(2P)s and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K(2P) family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus.

Multiple C-terminal tail Ca(2+)/CaMs regulate Ca(V)1.2 function but do not mediate channel dimerization.

Interactions between voltage-gated calcium channels (Ca(V)s) and calmodulin (CaM) modulate Ca(V) function. In this study, we report the structure of a Ca(2+)/CaM Ca(V)1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca(2+)/CaMs and two Ca(2+)/CaM-IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca(2+)/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes Ca(V)1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca(2+)/CaMs in the complex have different properties. Ca(2+)/CaM bound to the PreIQ C-region is labile, whereas Ca(2+)/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca(2+)/CaMs can bind the Ca(V)1.2 tail simultaneously and indicate a functional role for Ca(2+)/CaM at the C-region site.

Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins.

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (Na(V)Sp1) PD forms a stand-alone, ion selective pore (Na(V)Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na(V)Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na(V)Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca(V)Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na(V)s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na(V)Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na(V)Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

Expression, purification, and characterization of anuran saxiphilins using thermofluor, fluorescence polarization, and isothermal titration calorimetry.

Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).1.

Patch-clamp characterization of the MscS-like mechanosensitive channel from Silicibacter pomeroyi.

Based on sequence similarity, the sp7 gene product, MscSP, of the sulfur-compound-decomposing Gram-negative marine bacterium Silicibacter pomeroyi belongs to the family of MscS-type mechanosensitive channels. To investigate MscSP channel properties, we measured its response to membrane tension using the patch-clamp technique on either a heterologous expression system using giant spheroplasts of MJF465 Escherichia coli strain (devoid of mechanosensitive channels MscL, MscS, and MscK), or on purified MscSP protein reconstituted in azolectin liposomes. These experiments showed typical pressure-dependent gating properties of a stretch-activated channel with a current/voltage plot indicating a rectifying behavior and weak preference for anions similar to the MscS channel of E. coli. However, the MscSP channel exhibited functional differences with respect to conductance and desensitization behavior, with the most striking difference between the two channels being the lack of inactivation in MscSP compared with MscS. This seems to result from the fact that although MscSP has a Gly in an equivalent position to MscS (G113), a position that is critical for inactivation, MscSP has a Glu residue instead of an Asn in a position that was recently shown to allosterically influence MscS inactivation, N117. To our knowledge, this study describes the first electrophysiological characterization of an MscS-like channel from a marine bacterium belonging to sulfur-degrading α-proteobacteria.

Chemical informatics and target identification in a zebrafish phenotypic screen.

Target identification is a core challenge in chemical genetics. Here we use chemical similarity to computationally predict the targets of 586 compounds that were active in a zebrafish behavioral assay. Among 20 predictions tested, 11 compounds had activities ranging from 1 nM to 10,000 nM on the predicted targets. The roles of two of these targets were tested in the original zebrafish phenotype. Prediction of targets from chemotype is rapid and may be generally applicable.

Structural basis for CaVα2δ:gabapentin binding.

Gabapentinoid drugs for pain and anxiety act on the CaVα2δ-1 and CaVα2δ-2 subunits of high-voltage-activated calcium channels (CaV1s and CaV2s). Here we present the cryo-EM structure of the gabapentin-bound brain and cardiac CaV1.2/CaVß3/CaVα2δ-1 channel. The data reveal a binding pocket in the CaVα2δ-1 dCache1 domain that completely encapsulates gabapentin and define CaVα2δ isoform sequence variations that explain the gabapentin binding selectivity of CaVα2δ-1 and CaVα2δ-2.

Development of covalent chemogenetic K2P channel activators.

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (Covalent Activation of TREK family K+ channels to cLAmp Membrane Potential) that leverages the discovery of a site in the K2P modulator pocket that reacts with electrophile-bearing derivatives of a TREK subfamily small molecule activator, ML335, to activate the channel irreversibly. We show that the CATKLAMP strategy can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a new means to alter K2P channel activity that should facilitate studies both molecular and systems level studies of K2P function and enable the search for new K2P modulators.

Electrochemical coupling in the voltage-dependent phosphatase Ci-VSP.

In the voltage-sensing phosphatase Ci-VSP, a voltage-sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we found that the interdomain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays showed that the linker is also needed for activity in the isolated PD. We also identified a late step of VSD motion in the full-length protein that depends on the linker. Notably, we found that this VSD motion requires PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme.