From cryo-EM, multiple protein structures in one shot.
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Cryoelectron Microscopy↗
Biomedical subjects
Fred J Sigworth
Publications and source records attributed to Fred J Sigworth.
Using cryo-EM to measure the dipole potential of a lipid membrane.
The dipole potential of a lipid bilayer membrane accounts for its much larger permeability to anions than cations and affects the conformation and function of membrane proteins. The absolute value of the dipole potential has been very difficult to measure, although its value has been estimated to range from 200 to 1,000 mV from ion translocation rates, the surface potential of lipid monolayers, and molecular dynamics calculations. Here, a point charge probe method was used to investigate the dipole potentials of both ester and ether lipid membranes. The interactions between electrons and lipid molecules were recorded by phase-contrast imaging using cryo-EM. The magnitude and the profile of the dipole potential along the bilayer normal were obtained by subtracting the contribution of the atomic potential from the cryo-EM image intensity. The peak dipole potential was estimated to be 510 and 260 mV for diphytanoylphosphatidylcholine and diphytanylphosphatidylcholine, respectively.
Cryoelectron Microscopy↗
Software tools for macromolecular microscopy.
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Computational Biology↗
Pharmacological activation and inhibition of Slack (Slo2.2) channels.
The Slack (Sequence like a calcium-activated K channel) (Slo2.2) gene is abundantly expressed in the mammalian brain and encodes a sodium-activated K+ (KNa) channel. Although the specific roles of Slack channel subunits in neurons remain to be identified, they may play a role in the adaptation of firing rate and in protection against ischemic injury. In the present study, we have generated a stable cell line expressing the Slack channel, and have analyzed the pharmacological properties of these channels in these cells and in Xenopus oocytes. Two known blockers of KNa channels, bepridil and quinidine, inhibited Slack currents in a concentration-dependent manner and decreased channel activity in excised membrane patches. The inhibition by bepridil was potent, with an IC50 of 1.0 microM for inhibition of Slack currents in HEK cells. In contrast, bithionol was found to be a robust activator of Slack currents. When applied to the extracellular face of excised patches, bithionol rapidly induced a reversible increase in channel opening, suggesting that it acts on Slack channels relatively directly. These data establish an important early characterization of agents that modulate Slack channels, a process essential for the experimental manipulation of Slack currents in neurons.
Animals↗
Gating of acid-sensitive ion channel-1: release of Ca2+ block vs. allosteric mechanism.
The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at -60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.
Acid Sensing Ion Channels↗
Microfluidic system for planar patch clamp electrode arrays.
We present a microfluidic system integrated with disposable cell interface partitions for simultaneous patch clamp recordings. Glass-supported poly(dimethylsiloxane) (PDMS) partitions, having a 2 microm air-blown aperture, were reversibly sealed to a microfluidic system including PDMS channels with isolation valves and microfabricated Ag/AgCl electrodes. Gigaseal recordings from RBL-1 cells were obtained with a 24% success rate. Simultaneous whole cell recordings from valve-isolated electrodes were obtained.
Animals↗
Cryo-EM and single particles.
Cryoelectronmicroscopy is a method for the imaging of macromolecules in the electron microscope. It was originally developed to determine membrane protein structures from two-dimensional crystals, but more recently "single-particle" techniques have become powerful and popular. Three-dimensional reconstructions are obtained from sets of single-particle images by extensive computer processing; the methods are being applied to many macromolecular assemblies.
Animals↗
Generation of functional fluorescent BK channels by random insertion of GFP variants.
The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the alpha subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The "success rate" of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions.
Amino Acid Sequence↗
Microchip technology in ion-channel research.
The electrical activity of living cells can be monitored in various ways, but for the study of ion channels and the drugs that affect them, the patch-clamp techniques are the most sensitive. Recent developments in microfabricated patch-clamp electrodes are reviewed, and technical challenges for the future are discussed.
Action Potentials↗
An air-molding technique for fabricating PDMS planar patch-clamp electrodes.
We present a new technique for fabricating planar patch electrodes in the laboratory. Planar electrodes are micromolded using a micron-sized stream of air to define an aperture in the silicone elastomer, polydimethylsiloxane (PDMS). We have previously demonstrated that planar PDMS electrodes make excellent patch electrodes after surface modification. We demonstrate single-channel measurements of the rSlo channel in Xenopus oocytes and whole-cell measurements in CHO and RBL mammalian cell lines, using planar PDMS electrodes.
Air↗
Can Shaker potassium channels be locked in the deactivated state?
For structural studies it would be useful to constrain the voltage sensor of a voltage-gated channel in its deactivated state. Here we consider one Shaker potassium channel mutant and speculate about others that might allow the channel to remain deactivated at zero membrane potential. Ionic and gating currents of F370C Shaker, expressed in Xenopus oocytes, were recorded in patches with internal application of the methanethiosulfonate reagent MTSET. It appears that the voltage dependence of voltage sensor movement is strongly shifted by reaction with internal MTSET, such that the voltage sensors appear to remain deactivated even at positive potentials. A disadvantage of this construct is that the rate of modification of voltage sensors by MTSET is quite low, approximately 0.17 mM(-1).s(-1) at -80 mV, and is expected to be much lower at depolarized potentials.
Amino Acid Sequence↗
Slick (Slo2.1), a rapidly-gating sodium-activated potassium channel inhibited by ATP.
Neuronal stressors such as hypoxia and firing of action potentials at very high frequencies cause intracellular Na+ to rise and ATP to be consumed faster than it can be regenerated. We report the cloning of a gene encoding a K+ channel, Slick, and demonstrate that functionally it is a hybrid between two classes of K+ channels, Na+-activated (KNa) and ATP-sensitive (KATP) K+ channels. The Slick channel is activated by intracellular Na+ and Cl- and is inhibited by intracellular ATP. Slick is widely expressed in the CNS and is detected in heart. We identify a consensus ATP binding site near the C terminus of the channel that is required for ATP and its nonhydrolyzable analogs to reduce open probability. The convergence of Na+, Cl-, and ATP sensitivity in one channel may endow Slick with the ability to integrate multiple indicators of the metabolic state of a cell and to adjust electrical activity appropriately.
Adenosine Triphosphate↗
Structural biology: Life's transistors.
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Crystallography, X-Ray↗
Three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor at 24 A resolution.
We report here the first three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R). From cryo-electron microscopic images of purified receptors embedded in vitreous ice, a three-dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 A resolution structure of the IP(3)R takes the shape of an uneven dumbbell, and is approximately 170 A tall. Its larger end is bulky, with four arms protruding laterally by approximately 50 A and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is approximately 155 A. The smaller end, whose lateral dimension is approximately 100 A, has structural features indicative of the membrane-spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.
Animals↗
Patch clamp on a chip.
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Biophysical Phenomena↗
Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells.
The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.
Coated Materials, Biocompatible↗
Automatic particle selection: results of a comparative study.
Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.
Algorithms↗
Classical detection theory and the cryo-EM particle selection problem.
Particle selection is an essential but tedious step in the determination of macromolecular structures by single particle reconstruction. This paper presents an automatic, multi-reference particle detection scheme that is based on the classical matched filter principle. It makes use of a pre-whitening filter to standardize the noise, a reduced representation of the references by means of principal component analysis, and a statistic for distinguishing particles from image artifacts. Standardizing the noise allows the noise-induced false-positive frequency to be estimated, and also allows the distribution of the discrimination statistic to be calculated a priori. The method is demonstrated with an annotated dataset of cryo-EM images.
Algorithms↗