On the stochastic properties of bursts of single ion channel openings and of clusters of bursts

1982 ◽  
Vol 300 (1098) ◽  
pp. 1-59 ◽  
Keyword(s):  
Steady State ◽  
Ion Channels ◽  
Computer Program ◽  
Ion Channel ◽  
Energy Supply ◽  
Single Channel ◽  
Transition Probabilities ◽  
Transition Rates ◽  
Open Time ◽  
Stochastic Properties

Characteristics of observed bursts of single channel openings were derived recently for two particular ion channel mechanisms. In this paper these methods are generalized so that the observable characteristics of bursts can be calculated directly for any mechanism that has transition probabilities that are independent of time as long as the process is at equilibrium or is maintained in a steady state by an energy supply. General expressions are given for the distributions of the open time, the number of openings per burst, the total open time per burst, the gaps within and between bursts, and so on. With the aid of these general results a single computer program can be written that will provide numerical values for such distributions for postulated mechanism, given only the transition rates between the various states. The results are illustrated by a numerical example of a mechanism in which two agonist molecules can bind sequentially, and either singly or doubly occupied receptor ion channels may open. The analogous theory is also given for the case where bursts of channel openings are grouped into clusters; many of the results bear a close analogy with those found for simple bursts.

Stochastic properties of ion channel openings and bursts in a membrane patch that contains two channels: evidence concerning the number of channels present when a record containing only single openings is observed

1990 ◽  
Vol 240 (1299) ◽  
pp. 453-477 ◽  
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Rate Constants ◽  
Single Channel ◽  
Large Range ◽  
Active Channels ◽  
Exact Calculations ◽  
Stochastic Properties ◽  
Membrane Patch ◽  
Simultaneous Activity

If a single ion channel record is observed in which two ion channels are never simultaneously open, then it is often of interest to know whether the observations indeed arose from the activity of only one ion channel. This question can be answered if it is possible to calculate the distribution of the duration of runs of single openings in a membrane patch that contains two active channels. If the observed run of single openings is much longer than that expected for a patch with two channels it is likely that only one channel was active. An approximate method is presented for calculating the distribution of the duration of runs of single openings in a patch with two active channels; this method has the advantage that it can be calculated from observable quantities, and requires no knowledge of the details of the ion-channel mechanism or its rate constants. The accuracy of this approximation is tested by exact calculations of the properties of runs of single openings, and of single bursts, for two specific mechanisms and a large range of rate constants. The approximation is good in all cases in which openings occur singly, or in closely spaced bursts. If, as is common in practice, openings occur in clusters that are separated by long shut periods, then overlap of clusters from two different channels may be detected, if no double opening is produced, as a period in the middle of a cluster in which the probability of being open doubles. The results derived here can be applied to such a period to test whether it results from the simultaneous activity of two channels, rather than from a change in the properties of a single channel.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

On the stochastic properties of single ion channels

1981 ◽  
Vol 211 (1183) ◽  
pp. 205-235 ◽  
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
The State ◽  
Agonist Action ◽  
Agonist Concentration ◽  
Relaxation Experiments ◽  
Flow Through ◽  
Single Channel Currents ◽  
Stochastic Properties ◽  
Channel Currents

It is desirable to be able to predict, from a specified mechanism, the appearance of currents that flow through single ion channels ( a ) to enable interpretation of experiments in which single channel currents are observed, and ( b ) to allow physical meaning to be attached to the results observed in kinetic (noise and relaxation) experiments in which the aggregate of many single channel currents is observed. With this object, distributions (and their means) are derived for the length of the sojourn in any specified subset of states (e. g. all shut states). In general these are found to depend not only on the state in which the sojourn starts, but also on the state that immediately follows the sojourn. The methods described allow derivation of the distribution of, for example, ( a ) the number of openings, and total length of the burst of openings, that may occur during a single occupancy, and ( b ) the apparent gap between such bursts. The methods are illustrated by their application to two simple theories of agonist action. The Castillo-Katz (non-cooperative) mechanism predicts, for example, that the number of openings per occupancy, and the apparent burst length, are independent of agonist concentration whereas a simple cooperative mechanism predicts that both will increase with agonist concentration.

Ion channels and disease

2020 ◽  
pp. 246-255
Author(s):  
Frances Ashcroft ◽  
Paolo Tammaro
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Single Channel ◽  
Cell Types ◽  
Channel Structure ◽  
Channel Proteins ◽  
Channel Function ◽  
Ion Channel Proteins ◽  
Single Channel Currents ◽  
Channel Currents

Ion channels are membrane proteins that act as gated pathways for the movement of ions across cell membranes. They are found in both surface and intracellular membranes and play essential roles in the physiology of all cell types. An ever-increasing number of human diseases are now known to be caused by defects in ion channel function. To understand how ion channel defects give rise to disease, it is helpful to understand how the ion channel proteins work. This chapter therefore considers what is known of ion channel structure, explains the properties of the single ion channel, and shows how single-channel currents give rise to action potentials and synaptic potentials.

Membrane patches as ion channel probes for scanning ion conductance microscopy

2016 ◽  
Vol 193 ◽  
pp. 81-97 ◽  
Author(s):  
Wenqing Shi ◽  
Yuhan Zeng ◽  
Lushan Zhou ◽  
Yucheng Xiao ◽  
Theodore R. Cummins ◽  
...  
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Single Channel ◽  
Donor Cell ◽  
Bk Channels ◽  
Ion Conductance ◽  
Current Time ◽  
Scanning Ion Conductance Microscopy ◽  
Close Proximity ◽  
Membrane Patch

We describe dual-barrel ion channel probes (ICPs), which consist of an open barrel and a barrel with a membrane patch directly excised from a donor cell. When incorporated with scanning ion conductance microscopy (SICM), the open barrel (SICM barrel) serves to measure the distance-dependent ion current for non-invasive imaging and positioning of the probe in the same fashion of traditional SICM. The second barrel with the membrane patch supports ion channels of interest and was used to investigate ion channel activities. To demonstrate robust probe control with the dual-barrel ICP-SICM probe and verify that the two barrels are independently addressable, current–distance characteristics (approach curves) were obtained with the SICM barrel and simultaneous, current–time (I–T) traces were recorded with the ICP barrel. To study the influence that the distance between ligand-gated ion channels (i.e., large conductance Ca2+-activated K+ channels/BK channels) and the ligand source (i.e., Ca2+ source) has on channel activations, ion channel activities were recorded at two fixed probe–substrate distances (Dps) with the ICP barrel. The two fixed positions were determined from approach curves acquired with the SICM barrel. One position was defined as the “In-control” position, where the probe was in close proximity to the ligand source; the second position was defined as the “Far” position, where the probe was retracted far away from the ligand source. Our results confirm that channel activities increased dramatically with respect to both open channel probability and single channel current when the probe was near the ligand source, as opposed to when the probe was far away from the ligand source.

scholarly journals Homomeric GluA2(R) AMPA receptors can conduct when desensitized

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ian D. Coombs ◽  
David Soto ◽  
Thomas P. McGee ◽  
Matthew G. Gold ◽  
Mark Farrant ◽  
...  
Keyword(s):  
Steady State ◽  
Ion Channels ◽  
Ampa Receptors ◽  
Single Channel ◽  
Fluctuation Analysis ◽  
Single Channel Recording ◽  
Auxiliary Subunits ◽  
Steady State Current ◽  
The Brain ◽  
Silent State

Abstract Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors (AMPARs), which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we present evidence that homomeric Q/R-edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits (γ-2, γ-8 or GSG1L), generates large fractional steady-state currents and anomalous current-variance relationships. Our results from fluctuation analysis, single-channel recording, and kinetic modeling, suggest that the steady-state current is mediated predominantly by conducting desensitized receptors. When combined with crystallography this unique functional readout of a hitherto silent state enabled us to examine cross-linked cysteine mutants to probe the conformation of the desensitized ligand binding domain of functioning AMPAR complexes.

scholarly journals Homomeric Q/R edited AMPA receptors conduct when desensitized

2019 ◽  
Author(s):  
Ian D. Coombs ◽  
David Soto ◽  
Thomas P. McGee ◽  
Matthew G. Gold ◽  
Mark Farrant ◽  
...  
Keyword(s):  
Steady State ◽  
Ion Channels ◽  
Ampa Receptors ◽  
Single Channel ◽  
Fluctuation Analysis ◽  
Single Channel Recording ◽  
Steady State Current ◽  
The Brain ◽  
Surprising Finding ◽  
Silent State

Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors, which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we report the surprising finding that homomeric Q/R edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits (γ-2, γ-8 or GSG1L), generates large steady-state currents and anomalous current-variance relationships. Using fluctuation analysis, single-channel recording, and kinetic modeling we demonstrate that the steady-state current is mediated predominantly by ‘conducting desensitized’ receptors. When combined with crystallography this unique functional readout of a hith-erto silent state enabled us to examine cross-linked cysteine mutants to probe the conformation of the desensitized ligand binding domain of functioning AMPAR complexes within the plasma membrane.

scholarly journals Two-sided block of a dual-topology F− channel

2015 ◽  
Vol 112 (18) ◽  
pp. 5697-5701 ◽  
Author(s):  
Daniel L. Turman ◽  
Jacob T. Nathanson ◽  
Randy B. Stockbridge ◽  
Timothy O. Street ◽  
Christopher Miller
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Kinetic Analysis ◽  
Single Channel ◽  
Negative Cooperativity ◽  
Binding Assays ◽  
Single Channel Recordings ◽  
Phage Display Libraries ◽  
Acidic Environments ◽  
State Kinetic

The Fluc family is a set of small membrane proteins forming F−-specific electrodiffusive ion channels that rescue microorganisms from F− toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.

scholarly journals Unsupervised idealization of ion channel recordings by Minimum Description Length: Application to human PIEZO1-channels

2017 ◽  
Author(s):  
Radhakrishnan Gnanasambandam ◽  
Morten Schak Nielsen ◽  
Christopher Nicolai ◽  
Frederick Sachs ◽  
Johannes Pauli Hofgaard ◽  
...  
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Markov Models ◽  
Single Channel ◽  
Minimum Description Length ◽  
Simulated Data ◽  
Multiple Channels ◽  
Electrophysiological Recordings ◽  
Single Channel Currents ◽  
Channel Currents

AbstractResearchers can investigate the mechanistic and molecular basis of many physiological phenomena in cells by analyzing the fundamental properties of single ion channels. These analyses entail recording single channel currents and measuring current amplitudes and transition rates between conductance states. Since most electrophysiological recordings contain noise, the data analysis can proceed by idealizing the recordings to isolate the true currents from the noise. This de-noising can be accomplished with threshold crossing algorithms and Hidden Markov Models, but such procedures generally depend on inputs and supervision by the user, thus requiring some prior knowledge of underlying processes. Channels with unknown gating and/or functional sub-states and the presence in the recording of currents from uncorrelated background channels present substantial challenges to unsupervised analyses.Here we describe and characterize an idealization algorithm based on Rissanen’s Minimum Description Length (MDL) Principle. This method uses minimal assumptions and idealizes ion channel recordings without requiring a detailed user input or a priori assumptions about channel conductance and kinetics.. Furthermore, we demonstrate that correlation analysis of conductance steps can resolve properties of single ion channels in recordings contaminated by signals from multiple channels. We first validated our methods on simulated data defined with a range of different signal-to-noise levels, and then showed that our algorithm can recover channel currents and their substates from recordings with multiple channels, even under conditions of high noise. We then tested the MDL algorithm on real experimental data from human PIEZO1 channels and found that our method revealed the presence of substates with alternate conductances.

AMPA and Kainate Receptors

2020 ◽  
Author(s):  
G. Brent Dawe ◽  
Patricia M. G. E. Brown ◽  
Derek Bowie
Keyword(s):  
Ion Channel ◽  
Glutamate Receptor ◽  
Single Channel ◽  
Channel Gating ◽  
Regulatory Proteins ◽  
Kainate Receptors ◽  
Mammalian Brain ◽  
Receptor Field ◽  
Neuronal Excitation ◽  
Ion Channel Proteins

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate-type glutamate receptors (AMPARs and KARs) are dynamic ion channel proteins that govern neuronal excitation and signal transduction in the mammalian brain. The four AMPAR and five KAR subunits can heteromerize with other subfamily members to create several combinations of tetrameric channels with unique physiological and pharmacological properties. While both receptor classes are noted for their rapid, millisecond-scale channel gating in response to agonist binding, the intricate structural rearrangements underlying their function have only recently been elucidated. This chapter begins with a review of AMPAR and KAR nomenclature, topology, and rules of assembly. Subsequently, receptor gating properties are outlined for both single-channel and synaptic contexts. The structural biology of AMPAR and KAR proteins is also discussed at length, with particular focus on the ligand-binding domain, where allosteric regulation and alternative splicing work together to dictate gating behavior. Toward the end of the chapter there is an overview of several classes of auxiliary subunits, notably transmembrane AMPAR regulatory proteins and Neto proteins, which enhance native AMPAR and KAR expression and channel gating, respectively. Whether bringing an ion channel novice up to speed with glutamate receptor theory and terminology or providing a refresher for more seasoned biophysicists, there is much to appreciate in this summation of work from the glutamate receptor field.

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