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Voltage-dependent ion channels regulate the opening of their pores by sensing the membrane voltage. This process underlies the propagation of action potentials and other forms of electrical activity in cells. The voltage dependence of these channels is governed by the transmembrane displacement of the positive charged S4 helix within their voltage-sensor domains. We use cryo-electron microscopy to visualize this movement in the mammalian Eag voltage-dependent potassium channel in lipid membrane vesicles with a voltage difference across the membrane. Multiple structural configurations show that the applied electric field displaces S4 toward the cytoplasm by two helical turns, resulting in an extended interfacial helix near the inner membrane leaflet. The position of S4 in this down conformation is sterically incompatible with an open pore, thus explaining how movement of the voltage sensor at hyperpolarizing membrane voltages locks the pore shut in this kind of voltage-dependent K⁺ (Kv) channel. The structures solved in lipid bilayer vesicles detail the intricate interplay between Kv channels and membranes, from showing how arginines are stabilized deep within the membrane and near phospholipid headgroups, to demonstrating how the channel reshapes the inner leaflet of the membrane itself.

The voltage-gated potassium channel Eag1 is overexpressed in tumor cells from a range of cancers, and inhibiting Eag1 reduces tumor growth. Whicher and Mackinnon determined the structure of a mammalian Eag1 bound to the inhibitor calmodulin at 3.78 Å resolution (see the Perspective by Toombes and Swartz). The organization of the voltage-sensing and pore domains differs from that of other potassium channels, indicating that the gating mechanism is distinct. The structure also shows how the channel can be closed by a ligand, independently of the position of the voltage sensor. Science , this issue p. 664 ; see also p. 646 Structure of a potassium channel reveals the mechanism of the interplay between voltage-dependent and ligand gating. Voltage-gated potassium (K v ) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the single-particle cryo–electron microscopy structure of mammalian K v 10.1, or Eag1, bound to the channel inhibitor calmodulin, at 3.78 angstrom resolution. Unlike previous K v structures, the S4-S5 linker of Eag1 is a five-residue loop and the transmembrane segments are not domain swapped, which suggest an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4-S5 linker allow calmodulin to bind to the intracellular domains and to close the potassium pore, independent of voltage-sensor position. The structure reveals an alternative gating mechanism for K v channels and provides a template to further understand the gating properties of Eag1 and related channels.

Small-conductance Ca 2+ -activated K + (SK) channels are expressed throughout the nervous system and affect both the intrinsic excitability of neurons and synaptic transmission. An increase in the concentration of intracellular calcium opens the channels to conduct potassium across the cell membrane. Lee and MacKinnon report cryo–electron microscopy structures of human SK4-calmodulin channel complexes. Activation occurs when calcium binds to calmodulin, a protein with two lobes, known as C and N, separated by a flexible region. Each monomer in the channel tetramer binds constitutively to the C-lobe of calmodulin. The N-lobe of calmodulin is reasonably unconstrained until it binds calcium. With calcium bound, it then binds to the channel and induces conformational changes that open the pore. Science , this issue p. 508 Structural insights into how Ca 2+ -bound calmodulin activates the small-conductance Ca 2+ -activated K + channel for neuronal excitation are explored. Small-conductance Ca 2+ -activated K + (SK) channels mediate neuron excitability and are associated with synaptic transmission and plasticity. They also regulate immune responses and the size of blood cells. Activation of SK channels requires calmodulin (CaM), but how CaM binds and opens SK channels has been unclear. Here we report cryo–electron microscopy (cryo-EM) structures of a human SK4-CaM channel complex in closed and activated states at 3.4- and 3.5-angstrom resolution, respectively. Four CaM molecules bind to one channel tetramer. Each lobe of CaM serves a distinct function: The C-lobe binds to the channel constitutively, whereas the N-lobe interacts with the S4-S5 linker in a Ca 2+ -dependent manner. The S4-S5 linker, which contains two distinct helices, undergoes conformational changes upon CaM binding to open the channel pore. These structures reveal the gating mechanism of SK channels and provide a basis for understanding SK channel pharmacology.

Mechanosensitive ion channels convert external mechanical stimuli into electrochemical signals for critical processes including touch sensation, balance, and cardiovascular regulation. The best understood mechanosensitive channel, MscL, opens a wide pore, which accounts for mechanosensitive gating due to in-plane area expansion. Eukaryotic Piezo channels have a narrow pore and therefore must capture mechanical forces to control gating in another way. We present a cryo-EM structure of mouse Piezo1 in a closed conformation at 3.7Å-resolution. The channel is a triskelion with arms consisting of repeated arrays of 4-TM structural units surrounding a pore. Its shape deforms the membrane locally into a dome. We present a hypothesis in which the membrane deformation changes upon channel opening. Quantitatively, membrane tension will alter gating energetics in proportion to the change in projected area under the dome. This mechanism can account for highly sensitive mechanical gating in the setting of a narrow, cation-selective pore.

X-ray structures of the human TRAAK mechanosensitive potassium channel reveal how build-up of tension in the lipid membrane can convert the channel from a non-conducting wedge shape associated with an inserted lipid acyl chain that blocks the pore to an expanded cross-sectional shape that prevents lipid entry and thus permits ion conduction. Activation of the mechanosensitive TRAAK ion channel Mechanosensitive ion channels are responsible for sensing touch, hearing and pain. The mechanism by which mechanical force gates these channels is not known. This study presents the X-ray crystal structures of TRAAK potassium ion channels in conductive and non-conductive conformations. The activation of these channels in nociceptive sensory neurons regulates the noxious threshold for mechanical force in mice. The structures show that the long alkyl chain of a lipid physically blocks the pore in the wedge-shaped non-conductive state. A build-up of tension in the lipid membrane can convert the channel to the conductive state that has an expanded cross-sectional shape that prevents lipid entry and thus permits ion conduction. Activation of mechanosensitive ion channels by physical force underlies many physiological processes including the sensation of touch, hearing and pain 1 , 2 , 3 , 4 , 5 . TRAAK (also known as KCNK4) ion channels are neuronally expressed members of the two-pore domain K + (K2P) channel family and are mechanosensitive 6 . They are involved in controlling mechanical and temperature nociception in mice 7 . Mechanosensitivity of TRAAK is mediated directly through the lipid bilayer—it is a membrane-tension-gated channel 8 . However, the molecular mechanism of TRAAK channel gating and mechanosensitivity is unknown. Here we present crystal structures of TRAAK in conductive and non-conductive conformations defined by the presence of permeant ions along the conduction pathway. In the non-conductive state, a lipid acyl chain accesses the channel cavity through a 5 Å-wide lateral opening in the membrane inner leaflet and physically blocks ion passage. In the conductive state, rotation of a transmembrane helix (TM4) about a central hinge seals the intramembrane opening, preventing lipid block of the cavity and permitting ion entry. Additional rotation of a membrane interacting TM2–TM3 segment, unique to mechanosensitive K2Ps, against TM4 may further stabilize the conductive conformation. Comparison of the structures reveals a biophysical explanation for TRAAK mechanosensitivity—an expansion in cross-sectional area up to 2.7 nm 2 in the conductive state is expected to create a membrane-tension-dependent energy difference between conformations that promotes force activation. Our results show how tension of the lipid bilayer can be harnessed to control gating and mechanosensitivity of a eukaryotic ion channel.

CLC channels mediate passive Cl− conduction, while CLC transporters mediate active Cl− transport coupled to H+ transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its ‘glutamate gate’ residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl− conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl− at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl− affinity distinguish CLC channels and transporters. Channels and transporters are two classes of proteins that transport molecules and ions – collectively referred to as “substrates” – across cell membranes. Channels form a pore in the membrane and the substrates diffuse through passively. Transporters, on the other hand, actively pump substrates across a membrane, consuming energy in the process. Thus, channels and transporters work in distinct ways. Channels and transporters most often have unrelated structures, but there are rare examples of both existing within the same family of structurally similar proteins. CLC proteins, for example, include both chloride ion channels and transporters that pump chloride ions in one direction by harnessing the energy from hydrogen ions flowing in the other direction. It remains unclear why some CLC proteins work as channels while others are transporters, especially since the two seem indistinguishable on the basis of the order of their amino acids – the building blocks of all proteins. The conservation of the amino acid sequences implies they are structurally very similar. How then can different members perform such energetically distinct processes? Park and MacKinnon now show that the answer to this question serves as a reminder of how subtle nature can be. Indeed, while the structure of a human CLC channel (called CLC-1) is indeed similar to those of CLC transporters, one amino acid adopts a unique shape that explains why the protein cannot act as a transporter. This specific amino acid, a glutamate, is central to the exchange of chloride and hydrogen ions in CLC transporters. Park and MacKinnon show that its conformation in the CLC-1 channel stops this exchange, while leaving the pore open for the passive transport of chloride ions. Also, two other amino acids along the ion diffusion pathway in the CLC channel are smaller than their counterparts in CLC transporters, and so allow chloride ions to diffuse through more quickly. Lastly, Park and MacKinnon also note that channels do not require a wide pore: instead ions can still flow rapidly through a narrow pore if the chemical environment inside permits it. CLC proteins perform a number of important roles in humans, and mutations in CLC-encoding genes underlie numerous heritable diseases. It remains too early to know how this mechanistic study may or may not impact treatments, yet the findings will likely interest scientists working on ion conduction mechanisms and the evolution of molecular function.

G-protein-gated inward rectifier K + (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5 Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K + channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K + channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and intracellular Na + ions participate in multi-ligand regulation of GIRK channels. An X-ray structure and electrophysiological analysis of mammalian G-protein-gated inward rectifier potassium channel GIRK2 in complex with βγ reveals a pre-open channel structure consistent with channel activation by membrane delimited G-protein subunits. GIRK2–G-protein βγ dimer structure The activation of G-protein-coupled receptors (GPCRs) leads to the release of the G-protein subunits Gα and Gβγ from the intracellular surface of the GPCR. Gβγ can then bind to, and activate, the G-protein-gated inward rectifier K + (GIRK) channel, causing the channel pore to open. The opening of GIRK channels drives the membrane voltage towards the resting (Nernst) potential, which slows the rate of membrane depolarization. In this manuscript, Matthew Whorton and Roderick MacKinnon solve the X-ray crystal structure of a mammalian GIRK2 channel in the presence of the βγ G-protein subunits. Although the overall structure of the βγ G-protein subunits is essentially the same in the presence of Gα or GIRK, the structures of GIRK and GIRK–Gβγ are quite different. The structure also reveals how the signalling lipid PIP2 and intracellular Na + ions help to regulate the activity of GIRKs.

The Ca 2+ -activated K + channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca 2+ renders Slo1 central to processes that couple electrical signalling to Ca 2+ -mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca 2+ and Mg 2+ at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage-sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca 2+ -binding domain from a neighbouring subunit. Unique structural features of the Slo1 voltage sensor suggest that it undergoes different conformational changes than other known voltage sensors. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels. Two complementary studies present the full-length high-resolution structure of a Slo1 channel in the presence or absence of Ca 2+ ions, in which an unconventional allosteric voltage-sensing mechanism regulates the Ca 2+ sensor in addition to the voltage sensor’s direct action on the pore. Slo1 potassium channel structure and activity Dual activation by voltage and calcium ions makes Slo1/BK channels essential to processes that couple membrane electrical excitability and cellular calcium signalling, such as muscle contraction or neuronal communication. In two complementary studies, Roderick MacKinnon and colleagues present full-length structures for a Slo1 channel, either in the presence or the absence of Ca 2+ ions, suggesting an unconventional allosteric mechanism, whereby the voltage sensor regulates the Ca 2+ sensor instead of the channel's pore directly. These findings explain a large body of biochemical, genetic and physiological data, from both basic and clinical research.

Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K+ channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K+ (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the \"force-from-lipid\" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K+ channels.

Piezo1 is an ion channel that gates open when mechanical force is applied to a cell membrane, thus allowing cells to detect and respond to mechanical stimulation. Molecular structures of Piezo1 reveal a large ion channel with an unusually curved shape. This study analyzes how such a curved ion channel interacts energetically with the cell membrane. Through membrane mechanical calculations, we show that Piezo1 deforms the membrane shape outside the perimeter of the channel into a curved ‘membrane footprint’. This membrane footprint amplifies the sensitivity of Piezo1 to changes in membrane tension, rendering it exquisitely responsive. We assert that the shape of the Piezo channel is an elegant example of molecular form evolved to optimize a specific function, in this case tension sensitivity. Furthermore, the predicted influence of the membrane footprint on Piezo gating is consistent with the demonstrated importance of membrane-cytoskeletal attachments to Piezo gating.