Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events

Feng, Qicheng; Auerbach, Anthony; Sachs, Frederick

doi:10.1016/s0006-3495(96)79568-1

Cited by 417 publications

(503 citation statements)

References 31 publications

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“…The closed-channel current was manually set to zero, and the entire filtered dataset from a given patch was idealized with the segmental k means (SKM) algorithm of QuB to identify single-channel transitions and estimate conductance levels and event durations (Qin et al, 1996;Qin, 2004). Because our data appeared to contain many rapid transitions between several closely spaced open levels, we empirically tested different sampling rates and resolutions.…”

Section: Methodsmentioning

confidence: 99%

Structural and Single-Channel Results Indicate That the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

Zhang¹,

Cho²,

Lolis³

et al. 2008

J. Neurosci.

118

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At most excitatory central synapses, glutamate is released from presynaptic terminals and binds to postsynaptic AMPA receptors, initiating a series of conformational changes that result in ion channel opening. Efficient transmission at these synapses requires that glutamate binding to AMPA receptors results in rapid and near-synchronous opening of postsynaptic receptor channels. In addition, if the information encoded in the frequency of action potential discharge is to be transmitted faithfully, glutamate must dissociate from the receptor quickly, enabling the synapse to discriminate presynaptic action potentials that are spaced closely in time.The current view is that the efficacy of agonists is directly related to the extent to which ligand binding results in closure of the binding domain. For glutamate to dissociate from the receptor, however, the binding domain must open. Previously, we showed that mutations in glutamate receptor subunit 2 that should destabilize the closed conformation not only sped deactivation but also altered the relative efficacy of glutamate and quisqualate. Here we present x-ray crystallographic and single-channel data that support the conclusions that binding domain closing necessarily precedes channel opening and that the kinetics of conformational changes at the level of the binding domain importantly influence ion channel gating. Our findings suggest that the stability of the closed-cleft conformation has been tuned during evolution so that glutamate dissociates from the receptor as rapidly as possible but remains an efficacious agonist.

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Section: Methodsmentioning

confidence: 99%

Structural and Single-Channel Results Indicate That the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

Zhang¹,

Cho²,

Lolis³

et al. 2008

J. Neurosci.

118

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“…These instantaneous growth rates were uniformly high during obvious uncapped states and near zero during capped states. With these instantaneous growth rates as reporters of the true hidden barbed-end state (capped or uncapped), we used QuB kinetic analysis software, available on line (28,29), for Baum-Welch idealization and to estimate rate constants for capping and uncapping. We assumed a simple, second-order model for capping protein (C) binding to Mg-ATP-actin filament barbed ends (A n ) of…”

Section: Data Collection and Analysismentioning

confidence: 99%

Single Molecule Kinetic Analysis of Actin Filament Capping

Kuhn

Pollard

2007

Journal of Biological Chemistry

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We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 ؋ 10 6 M ؊1 s ؊1 and dissociation of 0.0003 s ؊1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ), PI(4,5)P 2 , and PI(3,4,5)P 3 , prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 ؋ 10 5 M ؊1 s ؊1 ) and a faster dissociation rate constant (0.004 s ؊1 ). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.The motility of animal cells depends on the coordinated assembly of actin filaments at the leading edge. Arp2/3 complex promotes actin polymerization by nucleating filaments that grow as branches from a mother filament (1, 2). Capping protein (CP, 3 called CapZ in muscle) appears to play an important role in generating the zone of short, highly branched actin filaments at the leading edge. CP is a heterodimer of structurally similar ␣ and ␤ subunits that form a mushroom-like structure with one flexible and one immobile COOH-terminal "tentacle" that bind the two terminal subunits of an actin filament (3). CP binds the barbed end of actin filaments tightly with a K d of 0.1-1 nM. Given an association rate constant of 3-4 M Ϫ1 s Ϫ1 and a cytoplasmic concentration of 1 M (4), CP should terminate the growth of actin filaments with a half-time of about 0.2 s after their nucleation. Terminating growth quickly keeps the filaments short, so that they do not buckle under the force of polymerization as they push the membrane forward.Vertebrate CP dissociates from barbed ends with a half-time of 30 min (4), far too slow for simple, spontaneous dissociation to be involved with the conversion of the network of short, branched filaments at the leading edge to longer, unbranched filaments toward the cell body. As CP prevents both dissociation of subunits from the barbed end and end-to-end filament annealing, some mechanism must accelerate the removal of CP from filament ends.Both lipids and proteins, such as Ena/VASP (5-7) and CARMIL (8), can inhibit the interaction of CP with actin filaments. Polyphosphatidylinositides (PPIs) bind CP and block its interaction with filaments both in vivo (9) and in vitro (10). Addition of PIP 2 to mixtures of capped filaments, CP, and actin monomers in vi...

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“…Segments of current traces showing single-channel activity were selected and idealized using the SKM algorithm of QuB (Qin et al 1996). Histograms of open-channel time distributions showed a single component and hence could be well fitted with single exponential curves using Igor Pro and their means were calculated (Fig.…”

Section: Dwell-time Analysismentioning

confidence: 99%

“…Rate-kinetics of the fast gating transitions (protonation/deprotonation) were fitted (MIL-algorithm) for each patch experiment separately with correction for missed events, imposing a dead-time of 90-100 ls (Qin et al 1996).…”

Section: Dwell-time Analysismentioning

confidence: 99%

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

et al. 2006

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Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12¢ position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12¢ lysines in the a, c and d subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK a values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in b or subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK a values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12¢ lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of a subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channel. Abbreviations nAChRNicotinic acetylcholine receptor TM1/TM2 First/second transmembrane segment GFP Green fluorescent protein

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Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events

Cited by 417 publications

References 31 publications

Structural and Single-Channel Results Indicate That the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

Structural and Single-Channel Results Indicate That the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

Single Molecule Kinetic Analysis of Actin Filament Capping

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

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