Publications by authors named "Roderick Mackinnon"

95 Publications

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

Proc Natl Acad Sci U S A 2022 03 14;119(12):e2114046119. Epub 2022 Mar 14.

Laboratory of Molecular Neurobiology and Biophysics, HHMI, The Rockefeller University, New York, NY ,10065.

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.
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http://dx.doi.org/10.1073/pnas.2114046119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8944589PMC
March 2022

Molecular structure of an open human K channel.

Proc Natl Acad Sci U S A 2021 11;118(48)

HHMI, The Rockefeller University, New York, NY 10065;

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. Here, we describe human pancreatic K (hK) structures with an open pore at 3.1- to 4.0-Å resolution using single-particle cryo-electron microscopy (cryo-EM). Pore opening is associated with coordinated structural changes within the ATP-binding site and the channel gate in Kir. Conformational changes in SUR are also observed, resulting in an area reduction of contact surfaces between SUR and Kir. We also observe that pancreatic hK exhibits the unique (among inward-rectifier channels) property of PIP-independent opening, which appears to be correlated with a docked cytoplasmic domain in the absence of PIP.
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http://dx.doi.org/10.1073/pnas.2112267118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8640745PMC
November 2021

Analysis of the mechanosensor channel functionality of TACAN.

Elife 2021 08 10;10. Epub 2021 Aug 10.

Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, New York, United States.

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 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-electron microscopy and observed that it is a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme A cofactor, confirmed by mass spectrometry. The TACAN protomer is related in three-dimensional structure to a fatty acid elongase, ELOVL7. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.
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http://dx.doi.org/10.7554/eLife.71188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376246PMC
August 2021

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

Elife 2020 08 26;9. Epub 2020 Aug 26.

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, New York, United States.

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. In the docked form the cytoplasmic domain, to which G binds, becomes accessible to the cytoplasmic membrane surface where G resides. Furthermore, PIP binding reshapes the G binding surface on the cytoplasmic domain, preparing it to receive G. We find that cardiac GIRK (GIRK1/4) can also exist in both extended and docked conformations. These findings lead us to conclude that PIP influences GIRK channels in a structurally similar manner to Kir2.2 channels. In Kir2.2 channels, the PIP-induced conformational changes open the pore. In GIRK channels, they prepare the channel for activation by G.
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http://dx.doi.org/10.7554/eLife.60552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556866PMC
August 2020

Structural Basis of Human KCNQ1 Modulation and Gating.

Cell 2020 01 26;180(2):340-347.e9. Epub 2019 Dec 26.

Laboratory of Molecular Neurobiology and Biophysics and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

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. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.
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http://dx.doi.org/10.1016/j.cell.2019.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083075PMC
January 2020

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

Elife 2019 12 9;8. Epub 2019 Dec 9.

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, New York, United States.

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. Four β4, each containing two transmembrane helices, encircle Slo1, contacting it through helical interactions inside the membrane. On the extracellular side, β4 forms a tetrameric crown over the pore. Structures with high and low Ca concentrations show that identical gating conformations occur in the absence and presence of β4, implying that β4 serves to modulate the relative stabilities of 'pre-existing' conformations rather than creating new ones. The effects of β4 on scorpion toxin inhibition kinetics are explained by the crown, which constrains access but does not prevent binding.
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http://dx.doi.org/10.7554/eLife.51409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934384PMC
December 2019

Voltage Sensor Movements during Hyperpolarization in the HCN Channel.

Cell 2019 12 28;179(7):1582-1589.e7. Epub 2019 Nov 28.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

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. We introduced a reversible, metal-mediated cross bridge into the voltage sensors to create the chemical equivalent of a hyperpolarized conformation and determined the structure using cryoelectron microscopy (cryo-EM). Unlike the depolarized HCN channel, the S4 helix is displaced toward the cytoplasm by two helical turns. Near the cytoplasm, the S4 helix breaks into two helices, one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels. These findings suggest a basis for allosteric communication between voltage sensors and the gate in this kind of channel. They also imply that voltage sensor movements are not the same in all voltage-gated channels.
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http://dx.doi.org/10.1016/j.cell.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911011PMC
December 2019

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

Elife 2019 11 22;8. Epub 2019 Nov 22.

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, New York, United States.

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. In this paper, using cryo-EM, we show that KvAP from has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.
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http://dx.doi.org/10.7554/eLife.52164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882556PMC
November 2019

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

Elife 2019 11 1;8. Epub 2019 Nov 1.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

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. 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. We speculate on why nodes of Ranvier contain a mechanosensitive K channel.
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http://dx.doi.org/10.7554/eLife.50403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824864PMC
November 2019

Regulation of Eag1 gating by its intracellular domains.

Elife 2019 09 6;8. Epub 2019 Sep 6.

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, New York, United States.

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. We demonstrate that the Eag1 intracellular domains are not required for voltage-dependent gating but likely interact with the VS to modulate gating. We identified specific interactions between the PAS, CNBHD, and VS that modulate voltage-dependent gating and provide evidence that VS movement destabilizes these interactions to promote channel opening. Additionally, mutation of these interactions renders Eag1 insensitive to calmodulin inhibition. The structure of the calmodulin insensitive mutant in a pre-open conformation suggests that channel opening may occur through a rotation of the intracellular domains and calmodulin may prevent this rotation by stabilizing interactions between the VS and intracellular domains. Intracellular domains likely play a similar modulatory role in voltage-dependent gating of the related K11-12 channels.
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http://dx.doi.org/10.7554/eLife.49188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731095PMC
September 2019

Force-induced conformational changes in PIEZO1.

Nature 2019 09 21;573(7773):230-234. Epub 2019 Aug 21.

Department of Anesthesiology, Weill Cornell Medicine, New York, NY, USA.

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. We also use high-speed atomic force microscopy to analyse the deformability of PIEZO1 under force in membranes on a mica surface, and show that PIEZO1 can be flattened reversibly into the membrane plane. By approximating the absolute force applied, we estimate a range of values for the mechanical spring constant of PIEZO1. Both methods of microscopy demonstrate that PIEZO1 can deform its shape towards a planar structure. This deformation could explain how lateral membrane tension can be converted into a conformation-dependent change in free energy to gate the PIEZO1 channel in response to mechanical perturbations.
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http://dx.doi.org/10.1038/s41586-019-1499-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7258172PMC
September 2019

Molecular basis of signaling specificity between GIRK channels and GPCRs.

Elife 2018 12 10;7. Epub 2018 Dec 10.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

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. K currents and bioluminescence resonance energy transfer between labelled G proteins and GIRK show that M2Rs catalyze Gβγ subunit release at higher rates than β2ARs, generating higher Gβγ concentrations that activate GIRK and regulate other targets of Gβγ. The higher rate of Gβγ release is attributable to a faster G protein coupled receptor - G protein trimer association rate in M2R compared to β2AR. Thus, a rate difference in a single kinetic step accounts for specificity.
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http://dx.doi.org/10.7554/eLife.42908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335053PMC
December 2018

Piezo's membrane footprint and its contribution to mechanosensitivity.

Elife 2018 11 27;7. Epub 2018 Nov 27.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

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.
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http://dx.doi.org/10.7554/eLife.41968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317911PMC
November 2018

Piezo1 forms a slowly-inactivating mechanosensory channel in mouse embryonic stem cells.

Elife 2018 08 22;7. Epub 2018 Aug 22.

Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, Howard Hughes Medical Institute, New York, United States.

Piezo1 is a mechanosensitive (MS) ion channel with characteristic fast-inactivation kinetics. We found a slowly-inactivating MS current in mouse embryonic stem (mES) cells and characterized it throughout their differentiation into motor-neurons to investigate its components. MS currents were large and slowly-inactivating in the stem-cell stage, and became smaller and faster-inactivating throughout the differentiation. We found that Piezo1 is expressed in mES cells, and its knockout abolishes MS currents, indicating that the slowly-inactivating current in mES cells is carried by Piezo1. To further investigate its slow inactivation in these cells, we cloned Piezo1 cDNA from mES cells and found that it displays fast-inactivation kinetics in heterologous expression, indicating that sources of modulation other than the aminoacid sequence determine its slow kinetics in mES cells. Finally, we report that Piezo1 knockout ES cells showed a reduced rate of proliferation but no significant differences in other markers of pluripotency and differentiation.
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http://dx.doi.org/10.7554/eLife.33149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128688PMC
August 2018

Structure of the CLC-1 chloride channel from .

Elife 2018 05 29;7. Epub 2018 May 29.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

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.
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http://dx.doi.org/10.7554/eLife.36629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019066PMC
May 2018

Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures.

Science 2018 05;360(6388):508-513

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA.

Small-conductance Ca-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-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.
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http://dx.doi.org/10.1126/science.aas9466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6241251PMC
May 2018

Molecular structure of human KATP in complex with ATP and ADP.

Elife 2017 12 29;6. Epub 2017 Dec 29.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

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 Mg and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg-ATP in the degenerate site and Mg-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 ADP's ability to override ATP inhibition.
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http://dx.doi.org/10.7554/eLife.32481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790381PMC
December 2017

Structure-based membrane dome mechanism for Piezo mechanosensitivity.

Elife 2017 12 12;6. Epub 2017 Dec 12.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

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.
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http://dx.doi.org/10.7554/eLife.33660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788504PMC
December 2017

Cryo-EM Structure of a KCNQ1/CaM Complex Reveals Insights into Congenital Long QT Syndrome.

Cell 2017 Jun;169(6):1042-1050.e9

Laboratory of Molecular Neurobiology and Biophysics and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (I) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an electrophysiological assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS.
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http://dx.doi.org/10.1016/j.cell.2017.05.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562354PMC
June 2017

Cryo-EM Structure of the Open Human Ether-à-go-go-Related K Channel hERG.

Cell 2017 04;169(3):422-430.e10

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

The human ether-à-go-go-related potassium channel (hERG, Kv11.1) is a voltage-dependent channel known for its role in repolarizing the cardiac action potential. hERG alteration by mutation or pharmacological inhibition produces Long QT syndrome and the lethal cardiac arrhythmia torsade de pointes. We have determined the molecular structure of hERG to 3.8 Å using cryo-electron microscopy. In this structure, the voltage sensors adopt a depolarized conformation, and the pore is open. The central cavity has an atypically small central volume surrounded by four deep hydrophobic pockets, which may explain hERG's unusual sensitivity to many drugs. A subtle structural feature of the hERG selectivity filter might correlate with its fast inactivation rate, which is key to hERG's role in cardiac action potential repolarization.
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http://dx.doi.org/10.1016/j.cell.2017.03.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484391PMC
April 2017

Structural Titration of Slo2.2, a Na-Dependent K Channel.

Cell 2017 01 19;168(3):390-399.e11. Epub 2017 Jan 19.

Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na-activated K channel, as a function of the Na concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na-dependent manner, without evidence of Na-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.
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http://dx.doi.org/10.1016/j.cell.2016.12.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382815PMC
January 2017

Structures of the Human HCN1 Hyperpolarization-Activated Channel.

Cell 2017 Jan 12;168(1-2):111-120.e11. Epub 2017 Jan 12.

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the control of rhythmic activity in cardiac and neuronal pacemaker cells. In HCN, the polarity of voltage dependence is uniquely reversed. Intracellular cyclic adenosine monophosphate (cAMP) levels tune the voltage response, enabling sympathetic nerve stimulation to increase the heart rate. We present cryo-electron microscopy structures of the human HCN channel in the absence and presence of cAMP at 3.5 Å resolution. HCN channels contain a K channel selectivity filter-forming sequence from which the amino acids create a unique structure that explains Na and K permeability. The voltage sensor adopts a depolarized conformation, and the pore is closed. An S4 helix of unprecedented length extends into the cytoplasm, contacts the C-linker, and twists the inner helical gate shut. cAMP binding rotates cytoplasmic domains to favor opening of the inner helical gate. These structures advance understanding of ion selectivity, reversed polarity gating, and cAMP regulation in HCN channels.
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http://dx.doi.org/10.1016/j.cell.2016.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496774PMC
January 2017

Structure of a CLC chloride ion channel by cryo-electron microscopy.

Nature 2017 01 21;541(7638):500-505. Epub 2016 Dec 21.

Laboratory of Molecular Neurobiology and Biophysics and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.

CLC proteins transport chloride (Cl) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl ions passively, whereas others are secondary active transporters that exchange two Cl ions for one H. The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture on the basis of sequence homology. Here we determined the structure of a bovine CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl/H transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl down its electrochemical gradient.
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http://dx.doi.org/10.1038/nature20812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576512PMC
January 2017

Structural basis for gating the high-conductance Ca-activated K channel.

Nature 2017 01 14;541(7635):52-57. Epub 2016 Dec 14.

Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10065, USA.

The precise control of an ion channel gate by environmental stimuli is crucial for the fulfilment of its biological role. The gate in Slo1 K channels is regulated by two separate stimuli, intracellular Ca concentration and membrane voltage. Slo1 is thus central to understanding the relationship between intracellular Ca and membrane excitability. Here we present the Slo1 structure from Aplysia californica in the absence of Ca and compare it with the Ca-bound channel. We show that Ca binding at two unique binding sites per subunit stabilizes an expanded conformation of the Ca sensor gating ring. These conformational changes are propagated from the gating ring to the pore through covalent linkers and through protein interfaces formed between the gating ring and the voltage sensors. The gating ring and the voltage sensors are directly connected through these interfaces, which allow membrane voltage to regulate gating of the pore by influencing the Ca sensors.
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http://dx.doi.org/10.1038/nature20775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513477PMC
January 2017

Cryo-EM structure of the open high-conductance Ca-activated K channel.

Nature 2017 01 14;541(7635):46-51. Epub 2016 Dec 14.

Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10065, USA.

The Ca-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 renders Slo1 central to processes that couple electrical signalling to Ca-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 and Mg 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-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.
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http://dx.doi.org/10.1038/nature20608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500982PMC
January 2017

Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism.

Science 2016 Aug;353(6300):664-9

Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA.

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.
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http://dx.doi.org/10.1126/science.aaf8070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477842PMC
August 2016

Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels.

Proc Natl Acad Sci U S A 2016 May 18;113(20):5748-53. Epub 2016 Apr 18.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065

K(+) channels, a superfamily of ∼80 members, control cell excitability, ion homeostasis, and many forms of cell signaling. Their malfunctions cause numerous diseases including neuronal disorders, cardiac arrhythmia, diabetes, and asthma. Here we present a novel liposome flux assay (LFA) that is applicable to most K(+) channels. It is robust, low cost, and high throughput. Using LFA, we performed small molecule screens on three different K(+) channels and identified new activators and inhibitors for biological research on channel function and for medicinal development. We further engineered a hERG (human ether-à-go-go-related gene) channel, which, when used in LFA, provides a highly sensitive (zero false negatives on 50 hERG-sensitive drugs) and highly specific (zero false positives on 50 hERG-insensitive drugs), low-cost hERG safety assay.
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http://dx.doi.org/10.1073/pnas.1602815113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878532PMC
May 2016

The GIRK1 subunit potentiates G protein activation of cardiac GIRK1/4 hetero-tetramers.

Elife 2016 04 13;5. Epub 2016 Apr 13.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, Rockefeller University, New York, United States.

G protein gated inward rectifier potassium (GIRK) channels are gated by direct binding of G protein beta-gamma subunits (Gβγ), signaling lipids, and intracellular Na(+). In cardiac pacemaker cells, hetero-tetramer GIRK1/4 channels and homo-tetramer GIRK4 channels play a central role in parasympathetic slowing of heart rate. It is known that the Na(+) binding site of the GIRK1 subunit is defective, but the functional difference between GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers remains unclear. Here, using purified proteins and the lipid bilayer system, we characterize Gβγ and Na(+) regulation of GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. We find in GIRK4 homo-tetramers that Na(+) binding increases Gβγ affinity and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na(+), but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na(+) permanently bound.
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http://dx.doi.org/10.7554/eLife.15750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866825PMC
April 2016

Cooperative regulation by G proteins and Na(+) of neuronal GIRK2 K(+) channels.

Elife 2016 04 13;5. Epub 2016 Apr 13.

Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, Rockefeller University, New York, United States.

G protein gated inward rectifier K(+) (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four Gβγ subunits bind cooperatively to open GIRK2, and that intracellular Na(+) - which enters neurons during action potentials - further amplifies opening mostly by increasing Gβγ affinity. A Na(+) amplification function is characterized and used to estimate the concentration of Gβγ subunits that appear in the membrane of mouse dopamine neurons when GABAB receptors are stimulated. We conclude that GIRK2, through its dual responsiveness to Gβγ and Na(+), mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity.
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http://dx.doi.org/10.7554/eLife.15751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866826PMC
April 2016

Cryo-electron microscopy structure of the Slo2.2 Na(+)-activated K(+) channel.

Nature 2015 Nov 5;527(7577):198-203. Epub 2015 Oct 5.

Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10065, USA.

Na(+)-activated K(+) channels are members of the Slo family of large conductance K(+) channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These channels fulfil a number of biological roles and have intriguing biophysical properties, including conductance levels that are ten times those of most other K(+) channels and gating sensitivity to intracellular Na(+). Here we present the structure of a complete Na(+)-activated K(+) channel, chicken Slo2.2, in the Na(+)-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 ångströms. The channel is composed of a large cytoplasmic gating ring, in which resides the Na(+)-binding site and a transmembrane domain that closely resembles voltage-gated K(+) channels. In the structure, the cytoplasmic domain adopts a closed conformation and the ion conduction pore is also closed. The structure reveals features that can explain the unusually high conductance of Slo channels and how contraction of the cytoplasmic gating ring closes the pore.
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http://dx.doi.org/10.1038/nature14958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886347PMC
November 2015
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