Stephen G. Brohawn

Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel.

Potassium Permeation Two–pore domain potassium (K2P) channels conduct K+ ions across the plasma membrane of eukaryotic cells. They help to maintain the cellular resting potential and their modulation can tune cellular excitability (see the Perspective by Poulsen and Nissen). Miller and Long (p. 432) describe a high-resolution crystal structure of the human K2P channel K2P1 (TWIK-1) and Brohawn et al. (p. 436) present a high-resolution structure of the lipid and mechanosensitive human channel TRAAK. In both structures an extracellular domain constricts the channel entrance so that K+ ions reach the selectivity filter through side portals. Openings in the transmembrane region expose the central cavity to the lipid bilayer and a helix is kinked so that its C-terminal part lies in the cytosol-membrane interface. The structural features explain K2P conductance and gating and give insight into how the channels are regulated by diverse stimuli. Structural features provide a basis for understanding gating and ion conduction of these channels. TRAAK channels, members of the two–pore domain K+ (potassium ion) channel family K2P, are expressed almost exclusively in the nervous system and control the resting membrane potential. Their gating is sensitive to polyunsaturated fatty acids, mechanical deformation of the membrane, and temperature changes. Physiologically, these channels appear to control the noxious input threshold for temperature and pressure sensitivity in dorsal root ganglia neurons. We present the crystal structure of human TRAAK at a resolution of 3.8 angstroms. The channel comprises two protomers, each containing two distinct pore domains, which create a two-fold symmetric K+ channel. The extracellular surface features a helical cap, 35 angstroms tall, that creates a bifurcated pore entryway and accounts for the insensitivity of two–pore domain K+ channels to inhibitory toxins. Two diagonally opposed gate-forming inner helices form membrane-interacting structures that may underlie this channel’s sensitivity to chemical and mechanical properties of the cell membrane.

Structural evidence for common ancestry of the nuclear pore complex and vesicle coats.

Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eightfold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the nucleoporin 85 (Nup85)⚫Seh1 structure, a module in the heptameric Nup84 complex, at 3.5 angstroms resolution. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identified interactions that define the organization of the Nup84 complex on the basis of comparison with vesicle coats and confirmed the sites by mutagenesis. We propose that the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice that provides docking sites for additional nucleoporins.

The Nuclear Pore Complex Has Entered the Atomic Age

Nuclear pore complexes (NPCs) perforate the nuclear envelope and represent the exclusive passageway into and out of the nucleus of the eukaryotic cell. Apart from their essential transport function, components of the NPC have important, direct roles in nuclear organization and in gene regulation. Because of its central role in cell biology, it is of considerable interest to determine the NPC structure at atomic resolution. The complexity of these large, 40-60 MDa protein assemblies has for decades limited such structural studies. More recently, exploiting the intrinsic modularity of the NPC, structural biologists are making progress toward understanding this nanomachine in molecular detail. Structures of building blocks of the stable, architectural scaffold of the NPC have been solved, and distinct models for their assembly proposed. Here we review the status of the field and lay out the challenges and the next steps toward a full understanding of the NPC at atomic resolution.

Mechanosensitivity is mediated directly by the lipid membrane in TRAAK and TREK1 K+ channels

Significance Mechanical force opens mechanosensitive ion channels in the cellular membrane to produce electrical signals that underlie sensation of touch, hearing, and other mechanical stimuli. An unanswered question is: How are mechanical forces transmitted to eukaryotic mechanosensitive channels in the membrane? We show that two mechanosensitive ion channels in eukaryotes, TRAAK (K2P4.1) and TREK1 (K2P2.1), are directly opened by mechanical force through the lipid membrane in the absence of all other cellular components. This finding extends the “force-from-lipid” paradigm established in bacterial channels to TRAAK and TREK1, eukaryotic mechanosensors. 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.

Domain-swapped chain connectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+ channel.

TRAAK (TWIK-related arachidonic acid-stimulated K+ channel, K2P4.1) K+ ion channels are expressed predominantly in the nervous system to control cellular resting membrane potential and are regulated by mechanical and chemical properties of the lipid membrane. TRAAK channels are twofold symmetric, which precludes a direct extension of gating mechanisms that close canonical fourfold symmetric K+ channels. We present the crystal structure of human TRAAK in complex with antibody antigen-binding fragments (Fabs) at 2.75-Å resolution. In contrast to a previous structure, this structure reveals a domain-swapped chain connectivity enabled by the helical cap that exchanges two opposing outer helices 180° around the channel. An unrelated conformational change of an inner helix seals a side opening to the membrane bilayer and is associated with structural changes around the K+-selectivity filter that may have implications for mechanosensitivity and gating of TRAAK channels.

The Structure of the Scaffold Nucleoporin Nup120 Reveals a New and Unexpected Domain Architecture

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous protein assemblies residing in circular openings in the nuclear envelope. The NPC is modular, with transient and stable components. The stable core is essentially built from two multiprotein complexes, the Y-shaped heptameric Nup84 complex and the Nic96 complex, arranged around an eightfold axis. We present the crystal structure of Nup120(1-757), one of the two short arms of the Y-shaped Nup84 complex. The protein adopts a compact oval shape built around a novel bipartite alpha-helical domain intimately integrated with a beta-propeller domain. The domain arrangement is substantially different from the Nup85*Seh1 complex, which forms the other short arm of the Y. With the data presented here, we establish that all three branches of the Y-shaped Nup84 complex are tightly connected by helical interactions and that the beta-propellers likely form interaction site(s) to neighboring complexes.

How ion channels sense mechanical force: insights from mechanosensitive K2P channels TRAAK, TREK1, and TREK2

The ability to sense and respond to mechanical forces is essential for life and cells have evolved a variety of systems to convert physical forces into cellular signals. Within this repertoire are the mechanosensitive ion channels, proteins that play critical roles in mechanosensation by transducing forces into ionic currents across cellular membranes. Understanding how these channels work, particularly in animals, remains a major focus of study. Here, I review the current understanding of force gating for a family of metazoan mechanosensitive ion channels, the two‐pore domain K+ channels (K2Ps) TRAAK, TREK1, and TREK2. Structural and functional insights have led to a physical model for mechanical activation of these channels. This model of force sensation by K2Ps is compared to force sensation by bacterial mechanosensitive ion channels MscL and MscS to highlight principles shared among these evolutionarily unrelated channels, as well as differences of potential functional relevance. Recent advances address fundamental questions and stimulate new ideas about these unique mechanosensors.

A lattice model of the nuclear pore complex.

The nuclear pore complex (NPC) is one of the largest protein machines in the cell and forms the sole conduit for nucleocytoplasmic transport in eukaryotes. The NPC is composed of an eightfold radially symmetric scaffold of architectural proteins that anchor a set of phenylalanine-glycine (FG) repeat proteins that form the transport barrier. As a step toward elucidating the molecular architecture of the NPC, we solved the structure of nucleoporin 85 (Nup85) in complex with Seh1, a module in the heptameric Nup84 subcomplex. We define a new tripartite protein element, the ancestral coatomer element ACE1, which Nup85 specifically shares with several other nucleoporins and vesicle coat proteins. We predicted and verified functional sites on nucleoporin ACE1 members based on analogy to ACE1 interactions that propagate the COPII vesicle coat. Thus, we provide the first experimental evidence for evolution of the NPC and vesicle coats from a common ancestor. We propose that the NPC structural scaffold, like vesicle coats, is a polygonal network composed of vertex and edge elements that forms a molecular lattice upon which additional nucleoporins assemble. Here we further discuss our findings and elaborate on our lattice model of the nuclear pore complex.