Biology subjects

MacKinnon, R.

Publications and source records attributed to MacKinnon, R..

15 recordsLinked to original sources

bioRxiv2026

PLCβs are recruited to the plasma membrane in macrophages by both Gβγ and Gαq

PLC{beta} enzymes cleave PIP2 from the plasma membrane, producing IP3 and DAG, which regulate intracellular Ca2+ levels and protein kinase C activity, respectively. They are regulated by GPCR signaling through the G proteins G{beta}{gamma} and Gq and have been shown to function as coincidence detectors for dual stimulation of Gq and Gi-coupled receptors via these G proteins. PLC{beta}s are aqueous-soluble enzymes, but partition onto the membrane surface to access their lipid substrate. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie G{beta}{gamma}-dependent regulation of PLC{beta} enzymes. Using macrophages as a model system, where PLC{beta} signaling is essential for responses to infection and tissue injury, we investigated the contribution of G{beta}{gamma}-dependent regulation and membrane recruitment of PLC{beta} in the context of endogenous signaling. By measuring Ca2+ mobilization, we demonstrate that both Gi and Gq-coupled receptors independently stimulate PLC{beta} activity. Using total internal reflection and stimulated emission depletion microscopy, we demonstrate that most of the PLC{beta}3 in the cell is localized away from the plasma membrane at rest but is rapidly recruited to the plasma membrane upon stimulation by both Gi and Gq-coupled receptors, illustrating that both G{beta}{gamma} and Gq recruit PLC{beta} to the plasma membrane. These results support an updated model for G protein-dependent regulation of PLC{beta} enzymes, where G{beta}{gamma}-induced regulation in the absence of Gq can occur and is apparently dictated by the local concentration of receptor, G proteins, and PLC{beta}. Significance StatementPLC{beta} enzymes are critical mediators of signal transduction with roles in neuronal, cardiac, and immunological signaling. Despite this importance, many aspects of their function and regulation remain poorly understood. PLC{beta}s are aqueous soluble but must partition onto the membrane surface to access their lipid substrate, which enables regulation at the partitioning step, the catalytic step, or both. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie PLC{beta} regulation by G{beta}{gamma} in a reconstituted system. Using macrophages as a model system for physiological signaling, we demonstrate here that G{beta}{gamma} can activate PLC{beta} via membrane recruitment in cells.

biophysics↗

bioRxiv2025

Spatial deconstruction of the plasma membrane

The plasma membrane of cells is known to be heterogeneous with regards to the spatial distribution of proteins and lipids, but the nature and origins of this heterogeneity are unclear. In this study we perform fluorescence microscopy on plasma membrane sheets and find that most proteins occur in protein-rich domains separated by intervening protein-poor regions. We show that the protein-rich domains and protein-poor regions are at least partially preserved in plasma membrane-derived vesicles that lack cytoskeletal elements, permitting their separation and isolation by density centrifugation. Compositional analysis by nuclear magnetic resonance and mass spectrometry shows that different proteins and lipids exhibit characteristic tendencies to partition into the protein-rich domains. This differential partitioning is correlated with the function of the different proteins, suggesting segregation based on cellular processes. Likewise, ordered lipids including cholesterol and sphingomyelin differentially segregate, being more abundant in the protein-rich domains. We propose that the collective assembly of certain proteins and lipids creates the heterogenous distribution of membrane components and the emergence of protein-rich domains. These domains could create distinct environments for the function and segregation of various membrane processes.

biophysics↗

bioRxiv2025

Lipid composition and mechanical force underlie multi-modal regulation of Piezo1 gating

Piezo1 ion channels are widely expressed cellular mechanosensors. They adopt an intrinsically curved shape when closed and are thought to open when mechanical forces applied to the membrane favor a more flattened conformation. By reconstituting Piezo1 channels into lipid vesicles, a flattened conformation has been determined, however, the ion conduction pore remained closed. In line with this observation, Piezo1 channels do not exhibit mechanical activation in the kind of lipids used in the structural studies. Here we show first that Piezo1 channels in cell-derived membranes retain functional mechanical gating, and second, that in cell-derived membranes they adopt a completely flattened disk shape associated with large conformational changes within and around the ion conduction pathway. These conformational changes occurring in cell-derived lipid membranes, suggest that mechanical force is necessary but insufficient, and that a specific membrane-derived cofactor apparently complements mechanical force to activate Piezo1.

biophysics↗

bioRxiv2024

Electric field-induced pore constriction in the human Kv2.1 channel

Gating in voltage-dependent ion channels is regulated by the transmembrane voltage. This form of regulation is enabled by voltage sensing domains (VSDs) that respond to transmembrane voltage differences by changing their conformation and exerting force on the pore to open or close it. Here we use cryogenic electron microscopy to study the neuronal Kv2.1 channel in lipid vesicles with and without a voltage difference across the membrane. Hyperpolarizing voltage differences displace the positively charged S4 helix in the voltage sensor by one helical turn ([~]5 [A]). When this displacement occurs, the S4 helix changes its contact with the pore at two different interfaces. When these changes are observed in fewer than four voltage sensors the pore remains open, but when they are observed in all four voltage sensors the pore constricts. The constriction occurs because the S4 helix, as it displaces inward, squeezes the right-handed helical bundle of pore lining S6 helices. A similar conformational change occurs upon hyperpolarization of the EAG1 channel. Therefore, while Kv2.1 and EAG1 are from distinct architectural classes of voltage-dependent ion channels, called domain-swapped and nondomain-swapped, the manner in which the voltage sensors gate their pores is very similar. Significance StatementOur ability to transmit signals across long distances rapidly - for example an instruction from the brain to the muscles in our fingers - depends on electrical impulses that travel along nerve cells. These electrical signals are mediated by membrane proteins called voltage-dependent ion channels. These channels have voltage sensors, which are domains that sense the voltage difference across the cell membrane and switch the channel on or off accordingly. Scientists discovered two architectural classes of voltage-dependent ion channels distinguished by the different ways the voltage sensors attach to the pore. This study shows that the two architectures are not very different after all because they both solve the problem of regulation of the pore by voltage sensors in the same way.

biochemistry↗

bioRxiv2024

Structure of the Flotillin Complex in a Native Membrane Environment

In this study we used cryo-electron 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 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 {beta}-strand, and coiled-coil domains that enable strong charge-charge inter-subunit interactions. Flotillin interacts with membranes at both ends; through its SPFH1 domains at the wide end and the C-terminus at the narrow end, facilitated by hydrophobic interactions and lipidation. The inward tilting of the SPFH domain, likely triggered by phosphorylation, suggests its role in membrane curvature induction, which could be connected to its proposed role in clathrin-independent endocytosis. The structure suggests a shared architecture across the family of SPFH proteins and will promote further research into Flotillins roles in cell biology. Significance statementIt is well known that many biochemical processes in cells must occur in localized regions. There are many different ideas about how cells keep processes localized. In this study we demonstrate that Flotillin1 and Flotillin2 co-assemble to form a large, truncated cone shaped cage whose wide end is always attached to a membrane surface and whose narrow end is sometimes attached to a separate membrane. The entire wall of the cage is without holes and is likely impervious even to small molecules, forming a diffusion barrier that can connect membrane systems. The Flotillin cage is thus well suited to isolate biochemical processes. Through membrane attachment, it also alters local membrane curvature, which could influence endocytic and mechanosensory processes.

biochemistry↗

bioRxiv2024

FBM: 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. This publication introduces the Freestanding-Bilayer Microscope (FBM), which combines the advantages of freestanding bilayers with single-particle tracking. The FBM, based on planar lipid bilayers, enables the study of IMP dynamics with single-molecule resolution and unconstrained diffusion. This paper benchmarks the FBM against total internal reflection fluorescence (TIRF) imaging on supported bilayers and is used here to estimate ion channel open probability and to examine the diffusion behavior of an ion channel in phase- separated bilayers. The FBM emerges as a powerful tool to examine membrane protein/lipid organization and dynamics to understand cell membrane processes.

biophysics↗

bioRxiv2023

The mechanism of Gαq regulation of PLCβ3-catalyzed PIP2 hydrolysis.

PLC{beta} enzymes cleave PIP2 producing IP3 and DAG. PIP2 modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca2+ levels and protein phosphorylation by protein kinase C, respectively. PLC{beta} enzymes are under the control of GPCR signaling through direct interactions with G proteins G{beta}{gamma} and Gq and have been shown to be coincidence detectors for dual stimulation of Gq and Gi coupled receptors. PLC{beta}s are aqueous-soluble cytoplasmic enzymes, but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that G{beta}{gamma} activates PLC{beta}3 by recruiting it to the membrane. Using these same methods, here we show that Gq increases the catalytic rate constant, kcat, of PLC{beta}3. Since stimulation of PLC{beta}3 by Gq depends on an autoinhibitory element (the X-Y linker), we propose that Gq produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the PLC{beta}3-Gq, and PLC{beta}3-G{beta}{gamma}(2)-Gq complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate PLC{beta}3 activity. The structures rationalize a finding in the enzyme assay, that co-stimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of PLC{beta}3 is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation. Significance StatementFor certain cellular signaling processes, the background activity of signaling enzymes must be minimal and stimulus-dependent activation robust. Nowhere is this truer than in signaling by PLC{beta}3, whose activity regulates intracellular Ca2+, phosphorylation by Protein Kinase C, and the activity of numerous ion channels and membrane receptors. In this study we show how PLC{beta}3 enzymes are regulated by two kinds of G proteins, G{beta}{gamma} and Gq. Enzyme activity studies and structures on membranes show how these G proteins act by separate, independent mechanisms, leading to a product rule of co-stimulation when they act together. The findings explain how cells achieve robust stimulation of PLC{beta}3 in the setting of very low background activity, properties essential to cell health and survival.

biophysics↗

bioRxiv2022

Gβγ Activates PIP2 Hydrolysis by Recruiting and Orienting PLCβ on the Membrane Surface

PLC{beta}s catalyze the hydrolysis of PIP2 into IP3 and DAG. PIP2 regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca2+ levels and activate PKC, respectively. PLC{beta}s are regulated by GPCRs through direct interaction with Gq and G{beta}{gamma}. This study addresses the mechanism by which G{beta}{gamma} activates PLC{beta}3. We show that PLC{beta}3 functions as a slow Michaelis-Menten enzyme (kcat~2 sec-1, KM~0.43 mol%) on membrane surfaces. Its partition coefficient (Kx~2.9 * 104) is such that only a small quantity of PLC{beta}3 exists in the membrane in the absence of G{beta}{gamma}. When G{beta}{gamma} is present, equilibrium binding (Keq~0.009 mol%) increases PLC{beta}3 in the membrane, increasing Vmax in proportion. Atomic structures on membrane vesicle surfaces show that two G{beta}{gamma} anchor PLC{beta}3 with its catalytic site oriented toward the membrane surface. This principle of activation explains rapid stimulated catalysis with low background catalysis.

biophysics↗

bioRxiv2022

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

Piezo1 is the stretch activated Ca2+ 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 towards the biconcave "dimple". Trajectories of diffusing Piezo1 molecules, which exhibit confined Brownian diffusion on short timescales and hopping on long timescales, also reflect a bias towards the dimple. This bias can be explained by "curvature coupling" between the intrinsic curvature of the Piezo dome and the curvature of the red blood cell membrane. Piezo1 does not form clusters with itself, nor does it co-localize with F-actin, Spectrin or the Gardos channel. Thus, Piezo1 exhibits the properties of a force-through-membrane sensor of curvature and lateral tension in the red blood cell.

biochemistry↗

bioRxiv2022

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

In a companion paper we show 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 (accompanying paper). Here we use these results to determine the force that Piezo exerts on the free membrane and, hence, that the free membrane exerts on Piezo, for a range of vesicle sizes. From vesicle shape measurements alone, we thus obtain a force-distortion relationship for Piezo, from which we deduce Piezos intrinsic radius of curvature, 42 {+/-} 12 nm, and bending stiffness, 18 {+/-} 2.1 kB T, in free-standing lipid bilayer membranes mimicking cell membranes. Applying these estimates to a spherical cap model of Piezo embedded in a lipid bilayer, we suggest that Piezos intrinsic curvature, surrounding membrane footprint, small stiffness, and large area are the key properties of Piezo that give rise to low-threshold, high-sensitivity mechanical gating.

biophysics↗

bioRxiv2022

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. MacKinnon, 2017). The curved shape of the Piezo dome is expected to deform the surrounding lipid bilayer membrane into a membrane footprint, which may serve to amplify Piezos sensitivity to applied forces (C. A. Haselwandter, R. MacKinnon, 2018). If Piezo proteins are embedded in lipid bilayer vesicles, the membrane shape deformations induced by the Piezo dome depend on the vesicle size. We employ here membrane elasticity theory to predict, with no free parameters, the shape of such Piezo vesicles outside the Piezo dome, and show that the predicted vesicle shapes agree quantitatively with the corresponding measured vesicle shapes obtained through cryo-electron tomography, for a range of vesicle sizes (Helfrich W. 1973). On this basis, we explore the coupling between Piezo and membrane shape, and demonstrate that the features of the Piezo dome affecting Piezos membrane footprint follow approximately a spherical cap geometry. Our work puts into place the foundation for deducing key elastic properties of the Piezo dome from membrane shape measurements and provides a general framework for quantifying how proteins deform bilayer membranes. ClassificationBiophysicss

biophysics↗

bioRxiv2021

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. Using cellular patch-recording methods we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods we found that TACAN, at high protein concentrations, produces non-selective, heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single particle cryo-EM and observe that it forms a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme-A co-factor, confirmed by mass spectrometry. The TACAN protomers are related in 3-dimensional structure to a fatty acid elongase, ELOVL. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.

biophysics↗

bioRxiv2021

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

Inward rectifier K+(Kir) channels regulate cell membrane potential. Different Kir channels respond to unique ligands, but all are regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Using planar lipid bilayers we show that Kir2.2 exhibits bursts of openings separated by long quiescent inter-burst periods. Increasing PI(4,5)P2 concentration shortens the Kir2.2 inter-burst duration and lengthens the burst duration without affecting dwell times within a burst. From this, we propose that burst and inter-burst durations correspond to the CTD-docked and CTD-undocked conformations observed in the presence and absence of PI(4,5)P2 in atomic structures. We also studied the effect of different phosphatidylinositol lipids on Kir2.2 activation and conclude that the 5 phosphate is essential to Kir2.2 pore opening. Other phosphatidylinositol lipids can compete with PI(4,5)P2 but cannot activate Kir2.2 without the 5 phosphate. PI(4)P, which is directly interconvertible to and from PI(4,5)P2, might thus be a regulator of Kir channels in the plasma membrane.

biophysics↗

bioRxiv2019

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 an all-nodes or no-nodes per axon fashion. TRAAK is not observed at the axon initial segment where action potentials are first generated. We used polyclonal antibodies, the TRAAK inhibitor RU2 and node clamp amplifiers to demonstrate the presence and functional properties of TRAAK in rat nerve fibers. TRAAK contributes to the leak K+ current in mammalian nerve fiber conduction by hyperpolarizing the resting membrane potential, thereby increasing Na+ channel availability for action potential propagation. Mechanical gating in TRAAK might serve a neuroprotective role by counteracting mechanically-induced ectopic action potentials. Alternatively, TRAAK may open in response to mechanical forces in the nodal membrane associated with depolarization during saltatory conduction and thereby contribute to repolarization of the node for subsequent spikes.

neuroscience↗

bioRxiv2017

Molecular structure of human KATP in complex with ATP and ADP

In many excitable cells KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K+ channel Kir6.2, in the presence of Mg2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 [A] and 5.6 [A], respectively. In both forms ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg2+-ATP in the degenerate site and Mg2+-ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form, and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADPs ability to override ATP inhibition.

biophysics↗