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was in close proximity to the ligand source; the second position was defined as the “Far” position, where the probe was retracted far away from the ligand source.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.

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 Characterization of Membrane Patch-Ion Channel Probes for Scanning Ion Conductance Microscopy

Small ◽  
2017 ◽  
Vol 14 (18) ◽  
pp. 1702945 ◽  
Author(s):  
Wenqing Shi ◽  
Yuhan Zeng ◽  
Cheng Zhu ◽  
Yucheng Xiao ◽  
Theodore R. Cummins ◽  
...  
Keyword(s):  
Ion Channel ◽  
Ion Conductance ◽  
Scanning Ion Conductance Microscopy ◽  
Membrane Patch

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.

scholarly journals Site-specific deacylation by ABHD17a controls BK channel splice variant activity

2020 ◽  
Vol 295 (49) ◽  
pp. 16487-16496 ◽  
Author(s):  
Heather McClafferty ◽  
Hamish Runciman ◽  
Michael J. Shipston
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Membrane Trafficking ◽  
Transmembrane Protein ◽  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Site Specific ◽  
And Function ◽  
A Site

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

scholarly journals Ion Channel Probes for Scanning Ion Conductance Microscopy

Langmuir ◽  
2014 ◽  
Vol 30 (50) ◽  
pp. 15351-15355 ◽  
Author(s):  
Yi Zhou ◽  
Leonard K. Bright ◽  
Wenqing Shi ◽  
Craig A. Aspinwall ◽  
Lane A. Baker
Keyword(s):  
Ion Channel ◽  
Ion Conductance ◽  
Scanning Ion Conductance Microscopy

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.

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.

Patch Fluorometry: Shedding New Light on Ion Channels

Physiology ◽  
2006 ◽  
Vol 21 (1) ◽  
pp. 6-12 ◽  
Author(s):  
Jie Zheng
Keyword(s):  
Ion Channels ◽  
Structure Function ◽  
Direct Observation ◽  
Single Channel ◽  
New Approach ◽  
Channel Protein ◽  
A Cell ◽  
Function Relationship ◽  
Free Membrane ◽  
Membrane Patch

Patch fluorometry has emerged as a new approach to the study of the structure-function relationship in membrane-embedded functional ion channels. Simultaneous fluorescent and electrical recordings are achieved from a small number of channels in a cell-free membrane patch, yielding high recording sensitivities. Further improvement of this approach should permit direct observation of the gating motion of a single-channel protein.

Abstract MP220: Identification And Functional Characterization Of Exosomal Bk Channels

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Shridhar Sanghvi ◽  
Julie A Dougherty ◽  
Parker Evans ◽  
Divya Sridharan ◽  
Devasena Ponnalagu ◽  
...  
Keyword(s):  
Ion Channel ◽  
Reperfusion Injury ◽  
Single Channel ◽  
Ischemia Reperfusion ◽  
Functional Characterization ◽  
Physiological Role ◽  
Bk Channels ◽  
Target Cells ◽  
Open Probability ◽  
Planar Bilayers

Extracellular vesicles (EVs) specifically exosomes are important in mediating intracellular communications, and are capable of transferring genetic information between cells. Exosomes are used as a drug delivery vehicle to carry cargo for targeted therapy and have emerged as one of the most promising candidate for treating cardiovascular diseases. Exosomes get packaged inside the cell, excise out to the extracellular environment, and deliver the cargo to the target cells. However, the precise mechanism of how exosomes handle the differential ionic environment and the physiological role of their ion channels is not determined. Given that potassium (K + ) ions has the largest gradient, we focused on identifying the presence and physiological relevance of K+ channels in exosomes. Using the in silico approach, several ion channel candidates were identified, the most prominent ion channel being large conductance Ca 2+ and voltage-activated potassium channel (BK). To record BK in exosomes, we incorporated planar bilayers and a novel electrophysiology approach called near field electrophysiology (NFE), as the canonical patch-clamp methods are not feasible due to the size of EVs. Our NFE indicates a presence of K + channels in intact exosomes and 45% of them are sensitive to IbTX. Since IbTX specifically blocks, BK channels, we estimated 2 functional channels (single-channel conductance of 300 pS with 50% open probability) in a single exosome. Plasma-derived exosomes from BK +/+ and BK -/- mice subjected to differential K + gradient indicated that functional BK channels exist in exosomes, and help in maintaining their structural integrity. Furthermore, plasma derived exosomes from BK +/+ mice showed cardioprotection from ischemia-reperfusion injury whereas exosomes from BK -/- mice did not. Thus, the presence of BK determines the packaging as well as cardioprotective function of exosomes. Overall, the study for the first time indicate a presence of functional ion channel (BK) in exosomes which plays a role in protecting cells and heart against ischemia and reperfusion injury.

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