Single‐Channel Recording

Jackson, Meyer B.

doi:10.1002/0471142301.ns0608s02

Cited by 5 publications

(7 citation statements)

References 57 publications

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“…17,18 In smaller cells, such as HEK cells, the patch clamp technique requires compensation for the resistance of the recording electrode and access to the cell, and even then, voltage artifacts are present when recording large currents, making peak current measurements unreliable. 19 Whatever the expression system, it is always difficult to clamp the V m when examining fast, large inward currents because during a depolarizing pulse the inward I Na is moving the potential in the same direction as the amplifier, creating unstable positive feedback. 20 These challenges in recording I Na are likely responsible for significant variability in published results, particularly regarding the magnitude of the late I Na , which relies on an accurate measure of the peak I Na .…”

Section: Mechanisms Of Inactivationmentioning

confidence: 99%

Mechanisms and models of cardiac sodium channel inactivation

et al. 2017

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Shortly after cardiac Na channels activate and initiate the action potential, inactivation ensues within milliseconds, attenuating the peak Na current, I and allowing the cell membrane to repolarize. A very limited number of Na channels that do not inactivate carry a persistent I, or late I. While late I is only a small fraction of peak magnitude, it significantly prolongs ventricular action potential duration, which predisposes patients to arrhythmia. Here, we review our current understanding of inactivation mechanisms, their regulation, and how they have been modeled computationally. Based on this body of work, we conclude that inactivation and its connection to late I would be best modeled with a "feet-on-the-door" approach where multiple channel components participate in determining inactivation and late I. This model reflects experimental findings showing that perturbation of many channel locations can destabilize inactivation and cause pathological late I.

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Section: Mechanisms Of Inactivationmentioning

confidence: 99%

Mechanisms and models of cardiac sodium channel inactivation

et al. 2017

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“…where k j represents the dissociation rate constant of the jth binding state and A j represents a weight applied upon the jth exponential function (j = 1, 2, …, m) 20,33 . The inverse of k j is the lifetime of PTEN membrane binding, τ j .…”

Section: Methodsmentioning

confidence: 99%

“…The dissociation curves in Fig. 2h were characterized by multiple rate constants and fitted by a two-component exponential function 20,33 . The dissociation rate constant for the shorter binding state (k 2 ) was decreased with increasing PI(4,5)P 2 density from 33.8 ± 6.9 s −1 at 1 mol% to 25.3 ± 6.2 s −1 at 10 mol%, while that for the longer binding state (k 1 ) was not significantly changed with a minimum of 3.7 ± 1.0 s −1 (10 mol%) and the maximum of 6.5 ± 4.8 s −1 (5 mol%) ( Fig.…”

Section: Pten-halomentioning

confidence: 99%

Single-molecule imaging of PI(4,5)P2 and PTEN in vitro reveals a positive feedback mechanism for PTEN membrane binding

et al. 2020

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PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P 3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P 2 , stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P 2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P 2 and thus the PI(4,5)P 2dependent stabilization. Single-molecule imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P 3 distribution to the anterior. These results suggest that PI(4,5)P 2 -mediated positive feedback and PTEN-induced PI(4,5)P 2 clustering may be important for anterior-posterior polarization.

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“…where I is the recorded current relative to baseline, t is time, i is the mean single-channel amplitude, and D is the duration of the ACh application (Jackson, 1998). No attempt was made to estimate P open for an individual channel because the total number of activatible channels in a patch could not be known with any degree of certainty, and because each patch served as its own control.…”

Section: Methodsmentioning

confidence: 99%

“…Absolute P open (NP open ) values were used as the primary measure of response to ACh for the outside-out patch clamp experiments in a manner analogous to the net charge measurements made from responses by receptors expressed in Xenopus oocytes. The NP open value was computed for an entire response to ACh, including the nonstationary phase of activation by

where I is the recorded current relative to baseline, t is time, i is the mean single-channel amplitude, and D is the duration of the ACh application ( Jackson, 1998 ). No attempt was made to estimate P open for an individual channel because the total number of activatible channels in a patch could not be known with any degree of certainty, and because each patch served as its own control.…”

Section: Methodsmentioning

confidence: 99%

The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

Williams

Stokes

Horenstein

et al. 2011

Journal of General Physiology

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We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists.

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Single‐Channel Recording

Cited by 5 publications

References 57 publications

Mechanisms and models of cardiac sodium channel inactivation

Mechanisms and models of cardiac sodium channel inactivation

Single-molecule imaging of PI(4,5)P2 and PTEN in vitro reveals a positive feedback mechanism for PTEN membrane binding

The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

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