Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution

Cell Calcium ◽  
2005 ◽  
Vol 37 (4) ◽  
pp. 283-299 ◽  
Author(s):  
Jianwei Shuai ◽  
Ian Parker
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Single Channel Recording ◽  
Individual Ion ◽  
Theoretical Considerations

Single-channel recording of ligand-gated ion channels

2007 ◽  
Vol 2 (11) ◽  
pp. 2826-2841 ◽  
Author(s):  
Martin Mortensen ◽  
Trevor G Smart
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Single Channel Recording ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

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 Single-channel Ca2+ imaging implicates Aβ1–42 amyloid pores in Alzheimer’s disease pathology

2011 ◽  
Vol 195 (3) ◽  
pp. 515-524 ◽  
Author(s):  
Angelo Demuro ◽  
Martin Smith ◽  
Ian Parker
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Ion Channels ◽  
Single Channel ◽  
Membrane Integrity ◽  
Cytosolic Calcium ◽  
Xenopus Laevis Oocytes ◽  
Permeable Membrane ◽  
Membrane Pores ◽  
Pathological Mechanism

Oligomeric forms of Aβ peptides are implicated in Alzheimer’s disease (AD) and disrupt membrane integrity, leading to cytosolic calcium (Ca2+) elevation. Proposed mechanisms by which Aβ mediates its effects include lipid destabilization, activation of native membrane channels, and aggregation of Aβ into Ca2+-permeable pores. We distinguished between these using total internal reflection fluorescence (TIRF) microscopy to image Ca2+ influx in Xenopus laevis oocytes. Aβ1–42 oligomers evoked single-channel Ca2+ fluorescence transients (SCCaFTs), which resembled those from classical ion channels but which were not attributable to endogenous oocyte channels. SCCaFTs displayed widely variable open probabilities (Po) and stepwise transitions among multiple amplitude levels reminiscent of subconductance levels of ion channels. The proportion of high Po, large amplitude SCCaFTs grew with time, suggesting that continued oligomer aggregation results in the formation of highly toxic pores. We conclude that formation of intrinsic Ca2+-permeable membrane pores is a major pathological mechanism in AD and introduce TIRF imaging for massively parallel single-channel studies of the incorporation, assembly, and properties of amyloidogenic oligomers.

Sign in / Sign up

Export Citation Format

Share Document