Structure of the flotillin complex in a native membrane environment.

In this study, we used cryoelectron microscopy to determine the structures of the Flotillin protein complex, part of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting of 22 pairs of Flotillin1 and Flotillin2 subunits, with a diameter of 32 nm at its wider end and 19 nm at its narrower end. Oligomerization is stabilized by the C terminus, which forms two helical layers linked by a β-strand, and coiled-coil domains that enable strong charge-charge intersubunit interactions.

Freestanding bilayer microscope for single-molecule imaging of membrane proteins.

Integral membrane proteins (IMPs) constitute a large fraction of organismal proteomes, playing fundamental roles in physiology and disease. Despite their importance, the mechanisms underlying dynamic features of IMPs, such as anomalous diffusion, protein-protein interactions, and protein clustering, remain largely unknown due to the high complexity of cell membrane environments. Available methods for in vitro studies are insufficient to study IMP dynamics systematically.

The mechanism of regulation of -catalyzed hydrolysis.

() enzymes cleave phosphatidylinositol 4,5-bisphosphate ( producing and (diacylglycerol). modulates the function of many ion channels, while and regulate intracellular Ca levels and protein phosphorylation by protein kinase C, respectively. enzymes are under the control of G protein coupled receptor signaling through direct interactions with G proteins and and have been shown to be coincidence detectors for dual stimulation of and -coupled receptors.

The mechanism of regulation of -catalyzed hydrolysis.

Unlabelled: enzymes cleave producing IP3 and DAG. modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca levels and protein phosphorylation by protein kinase C, respectively. enzymes are under the control of GPCR signaling through direct interactions with G proteins and and have been shown to be coincidence detectors for dual stimulation of and G coupled receptors.

The membrane electric field regulates the PIP-binding site to gate the KCNQ1 channel.

Voltage-dependent ion channels underlie the propagation of action potentials and other forms of electrical activity in cells. In these proteins, voltage sensor domains (VSDs) regulate opening and closing of the pore through the displacement of their positive-charged S4 helix in response to the membrane voltage. The movement of S4 at hyperpolarizing membrane voltages in some channels is thought to directly clamp the pore shut through the S4-S5 linker helix.

activates hydrolysis by recruiting and orienting on the membrane surface.

catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate [Formula: see text] into [Formula: see text] [Formula: see text] and [Formula: see text]  [Formula: see text]. [Formula: see text] regulates the activity of many membrane proteins, while and lead to increased intracellular Ca levels and activate protein kinase C, respectively. are regulated by G protein-coupled receptors through direct interaction with [Formula: see text] and [Formula: see text] and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate.

Membrane protein isolation and structure determination in cell-derived membrane vesicles.

Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.

Freestanding lipid bilayer tensiometer for the study of mechanosensitive ion channels.

Mechanical forces modify the cell membrane potential by opening mechanosensitive ion channels. We report the design and construction of a lipid bilayer tensiometer to study channels that respond to lateral membrane tension, [Formula: see text] , in the range 0.2 to 1.

Piezo1 as a force-through-membrane sensor in red blood cells.

Piezo1 is the stretch activated Ca channel in red blood cells that mediates homeostatic volume control. Here, we study the organization of Piezo1 in red blood cells using a combination of super-resolution microscopy techniques and electron microscopy. Piezo1 adopts a non-uniform distribution on the red blood cell surface, with a bias toward the biconcave 'dimple'.

Voltage-sensor movements in the Eag Kv channel under an applied electric field.

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.

Structural and functional analyses of a GPCR-inhibited ion channel TRPM3.

G-protein coupled receptors (GPCRs) govern the physiological response to stimuli by modulating the activity of downstream effectors, including ion channels. TRPM3 is an ion channel inhibited by GPCRs through direct interaction with G protein (Gβγ) released upon their activation. This GPCR-TRPM3 signaling pathway contributes to the analgesic effect of morphine.

Elastic properties and shape of the Piezo dome underlying its mechanosensory function.

We show in the companion paper that the free membrane shape of lipid bilayer vesicles containing the mechanosensitive ion channel Piezo can be predicted, with no free parameters, from membrane elasticity theory together with measurements of the protein geometry and vesicle size [C. A. Haselwandter, Y.

Quantitative prediction and measurement of Piezo's membrane footprint.

Piezo proteins are mechanosensitive ion channels that can locally curve the membrane into a dome shape [Y. R. Guo, R.

Correlation between structure and function in phosphatidylinositol lipid-dependent Kir2.2 gating.

SignificancePhosphatidylinositol 4,5-bisphosphate (PI(4,5)P) levels regulate cell membrane voltage by gluing two halves of a K channel together and opening the pore. PI(4)P competes with this process. Because both of these lipids are relatively abundant in the plasma membrane and are directly interconvertible through the action of specific enzymes, they may function together to regulate channel activity.

Molecular structure of an open human K channel.

K channels are metabolic sensors that translate intracellular ATP/ADP balance into membrane excitability. The molecular composition of K includes an inward-rectifier potassium channel (Kir) and an ABC transporter-like sulfonylurea receptor (SUR). Although structures of K have been determined in many conformations, in all cases, the pore in Kir is closed.

Analysis of the mechanosensor channel functionality of TACAN.

Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel.

Cryo-EM analysis of PIP regulation in mammalian GIRK channels.

G-protein-gated inward rectifier potassium (GIRK) channels are regulated by G proteins and PIP. Here, using cryo-EM single particle analysis we describe the equilibrium ensemble of structures of neuronal GIRK2 as a function of the C8-PIP concentration. We find that PIP shifts the equilibrium between two distinguishable structures of neuronal GIRK (GIRK2), extended and docked, towards the docked form.

Structural Basis of Human KCNQ1 Modulation and Gating.

KCNQ1, also known as Kv7.1, is a voltage-dependent K channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5.

Molecular structures of the human Slo1 K channel in complex with β4.

Slo1 is a Ca- and voltage-activated K channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slo1 is regulated by auxiliary proteins that confer tissue-specific gating and pharmacological properties. This study presents cryo-EM structures of Slo1 in complex with the auxiliary protein, β4.

Voltage Sensor Movements during Hyperpolarization in the HCN Channel.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a voltage-gated cation channel that mediates neuronal and cardiac pacemaker activity. The HCN channel exhibits reversed voltage dependence, meaning it closes with depolarization and opens with hyperpolarization. Different from Na, Ca, and Kv1-Kv7 channels, the HCN channel does not have domain-swapped voltage sensors.

Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology.

Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels.

The mechanosensitive ion channel TRAAK is localized to the mammalian node of Ranvier.

TRAAK is a membrane tension-activated K channel that has been associated through behavioral studies to mechanical nociception. We used specific monoclonal antibodies in mice to show that TRAAK is localized exclusively to nodes of Ranvier, the action potential propagating elements of myelinated nerve fibers. Approximately 80 percent of myelinated nerve fibers throughout the central and peripheral nervous system contain TRAAK in what is likely an all-nodes or no-nodes per axon fashion.

Regulation of Eag1 gating by its intracellular domains.

Voltage-gated potassium channels (Ks) are gated by transmembrane voltage sensors (VS) that move in response to changes in membrane voltage. K10.1 or Eag1 also has three intracellular domains: PAS, C-linker, and CNBHD.

Force-induced conformational changes in PIEZO1.

PIEZO1 is a mechanosensitive channel that converts applied force into electrical signals. Partial molecular structures show that PIEZO1 is a bowl-shaped trimer with extended arms. Here we use cryo-electron microscopy to show that PIEZO1 adopts different degrees of curvature in lipid vesicles of different sizes.

Molecular basis of signaling specificity between GIRK channels and GPCRs.

Stimulated muscarinic acetylcholine receptors (M2Rs) release Gβγ subunits, which slow heart rate by activating a G protein-gated K channel (GIRK). Stimulated β2 adrenergic receptors (β2ARs) also release Gβγ subunits, but GIRK is not activated. This study addresses the mechanism underlying this specificity of GIRK activation by M2Rs.