Mean Open Duration Research Articles

Microscopic mechanism of PIEZO1 activation by pressure-induced membrane stretch.

Mechanosensitive PIEZO1 ion channels open in response to membrane stretch. Yet, the underlying microscopic mechanism of this activation remains unknown. To probe this mechanism, we used cell-attached pressure-clamp recordings to measure single channel currents at different steady-state negative pipette pressures, spanning the full range of the channel's pressure sensitivity. Pressure-dependent activation occurs through a sharp reduction of the mean shut duration and through a moderate increase of the mean open duration. Across all tested pressures, the distribution of open and shut dwell times best follows sums of two and three exponential components, respectively. As the magnitude of the pressure stimulus increases, the time constants of most of these exponential components gradually change, in opposite directions for open and shut dwell times, and to a similar extent. In addition, while the relative amplitudes of fast and slow components remain unchanged for open intervals, they fully reverse for shut intervals, further reducing the mean shut duration. Using two-dimensional dwell time analysis, Markov-chain modeling, and simulations, we identified a minimal five-states model which recapitulates essential characteristics of single channel data, including microscopic reversibility, correlations between adjacent open and shut intervals, and asymmetric modulation of dwell times by pressure. This study identifies a microscopic mechanism for the activation of PIEZO1 channels by pressure-induced membrane stretch and deepens our fundamental understanding of mechanotransduction by a vertebrate member of the PIEZO channel family.

Open abdomen management for severe peritonitis in elderly. Results from the prospective International Register of Open Abdomen (IROA): Cohort study.

Analyzing the data of the International Register of Open Abdomen (IROA), the feasibility of open abdomen treatment has been demonstrated at every age. This new analysis on the IROA database investigates the risk factors for mortality in elderly patients treated with open abdomen for intra-abdominal infection. Data were derived from the IROA, a prospective observational international cohort study that enrolled patients treated with open abdomen worldwide. A univariate analysis of potential risk factors was performed. Inclusion criteria were patients older than 65 years and treated with open abdomen for intra-abdominal infection. End point was overall mortality, calculated within 30 days after open abdomen management, after 1-month and 1-year follow-up. A total of 116 patients was analyzed with mean age of 76 ± 7 years. Definitive closure was achieved in 93 patients (93/116, 80.2%) for a mean open abdomen duration of 5.0 ± 5.0 days. Complicated patients were 101 (101/116, 87.1%) for a total of 201 complications. Overall, 62 out of 116 patients (53.4%) died: 23 patients (23/62, 37.1%) during open abdomen management, 29 patients (46.8%) within 30 days after abdominal closure, 9 patients (14.5%) after 1-month follow-up, and 1 patient (1.6%) after 1-year follow-up. Age did not affect mortality (75 ± 6 years in alive patients versus 77 ± 7 years in dead patients, p = 0.773). Definitive abdominal closure was the most important factor to prevent mortality. This study confirmed that age alone cannot be considered a determinant for death, even in elderly patients managed with open abdomen for severe intra-abdominal infection.

Open abdomen and age; results from IROA (International Register of Open Abdomen)

IntroductionNo definitive data exist regarding epidemiology and outcomes of patients treated with open abdomen (OA) linked to age. MethodsThis is a prospective, observational, cohort study that includes patients treated with OA. Patients were divided into 4 age groups: group 1: 16–40 y.o.; group 2: 41–60 y.o.; group 3:61–80 y.o.; group 4:> 80 y.o. Results760 patients were enrolled. Average age was 60 ± 18aa; 57.2% were males. Most frequent indication was peritonitis (50.9%). Mean open duration of open abdomen was8±18 days. Definitive closure was reached in 81.1% of patients. Complications were recorded in 84.8% of patients with significant differences between groups (p = 0.001). Overall mortality was41.2% with significant differences between groups (p < 0.001) (group 1 25.6%, group 2 36.1%, group 3 44.5%, group 4 67.1%) 1 month follow-up mortality post-OA was 9.2% and 1 year follow-up mortality was 11.6%; ConclusionsOpen abdomen treatment is feasible at every age. Diseases requiring treatment with OA are differently distributed in relation to the different age groups. Advanced age has negative effect on complications and mortality rate.

Mapping Structural Elements to NMDA Receptor Activation Steps

NMDA receptors are heterotetrameric glutamate-activated ionotropic receptors that mediate excitatory synaptic transmission in the brain. Numerous NMDA receptor subunit mutations are associated with epilepsy and several have been identified as causal factors. Single-molecule electrophysiological recordings reveal that NMDA receptors explore many functional configurations. However, our ability to map structural changes to each of these functional configurations has been limited. Here, we integrate single-molecule recordings of GluN1-1a/GluN2A receptors with computational modeling of receptor activation to determine structural elements necessary for receptor activation. We found using single-channel recordings of disease-associated mutation, GluN1-1a I642L (GluN1-1aI642L) reduced receptor open probability within bursts (wild-type: 0.72 ± 0.04; GluN1-1aI642L: 0.04 ± 0.06), mean open duration (wild-type: 4.34 ± 0.11 msec; GluN1-1aI642L: 1.10 ± 0.09 msec), and mean closed duration within bursts (wild-type: 3.21 ± 0.51 msec; GluN1-1aI642L: 11.74 ± 1.20 msec). To determine the structural mechanism by which this disease mutation perturbs channel function, we used GluN1/GluN2B crystal structures to build a homology model of GluN1/GluN2A lacking cytoplasmic and amino terminal domains. Using this model, we performed targeted molecular dynamics simulations of the closed receptor into a putative “open” conformation. We found that residue GluN1 I642 forms additional atomic contacts with L550 on the neighboring GluN2A during this transition. We performed mutant cycle analysis on these pair of residues by recording single-channel currents through GluN1-1a/GluN2A, GluN1-1AI642L/GluN2A, GluN1-1a/GluN2AL550I, or GluN1-1AI642L/GluN2AL550I. Using kinetic modeling of these currents to measure the free energy change associated with each transition step in the receptor activation pathway, we found that the strongest coupling between GluN1 I642 and GluN2A L550 occurs transitions between states C3 to C2 and O1 to O2. Our approach provides a tool by which we can begin to map precise structural elements necessary for each transition step in receptor function.

Correction to 'Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels'.

Open AccessMoreSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail Cite this article Proks Peter, Puljung Michael C., Vedovato Natascia, Sachse Gregor, Mulvaney Rachel and Ashcroft Frances M. 2016Correction to ‘Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels’Phil. Trans. R. Soc. B3712016033020160330http://doi.org/10.1098/rstb.2016.0330SectionOpen AccessCorrectionCorrection to ‘Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels’ Peter Proks Peter Proks Google Scholar Find this author on PubMed Search for more papers by this author , Michael C. Puljung Michael C. Puljung Google Scholar Find this author on PubMed Search for more papers by this author , Natascia Vedovato Natascia Vedovato Google Scholar Find this author on PubMed Search for more papers by this author , Gregor Sachse Gregor Sachse Google Scholar Find this author on PubMed Search for more papers by this author , Rachel Mulvaney Rachel Mulvaney Google Scholar Find this author on PubMed Search for more papers by this author and Frances M. Ashcroft Frances M. Ashcroft Google Scholar Find this author on PubMed Search for more papers by this author Peter Proks Peter Proks Google Scholar Find this author on PubMed Search for more papers by this author , Michael C. Puljung Michael C. Puljung Google Scholar Find this author on PubMed Search for more papers by this author , Natascia Vedovato Natascia Vedovato Google Scholar Find this author on PubMed Search for more papers by this author , Gregor Sachse Gregor Sachse Google Scholar Find this author on PubMed Search for more papers by this author , Rachel Mulvaney Rachel Mulvaney Google Scholar Find this author on PubMed Search for more papers by this author and Frances M. Ashcroft Frances M. Ashcroft Google Scholar Find this author on PubMed Search for more papers by this author Published:05 September 2016https://doi.org/10.1098/rstb.2016.0330This article corrects the followingReview ArticleRunning out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channelshttps://doi.org/10.1098/rstb.2015.0426 Peter Proks, Michael C. Puljung, Natascia Vedovato, Gregor Sachse, Rachel Mulvaney and Frances M. Ashcroft volume 371issue 1700Philosophical Transactions of the Royal Society B: Biological Sciences05 August 2016Phil. Trans. R. Soc. B371, 20150426 (2016; Published 4 July 2016) (doi:10.1098/rstb.2015.0426)The x-axis labels in figure 2b are incorrect. The corrected figure 2 is given below. Figure 2. Effect of rundown on single-channel KATP channel properties. (a) Representative 1 min recording of single Kir6.2-G334D/SUR1 channels at −60 mV in the cell-attached configuration (a, Po = 0.83) and after 5-min after patch excision (b, Po = 0.22). (b) Distributions of channel open times (i), closed times (ii) and burst duration (iii) before (pale grey bars) and after (dark grey bars) rundown. The distributions were fitted with probability density functions that gave the following values for the individual components. Before rundown: mean open time, 2.7 ms; three apparent closed states with mean values of 0.3 ms (98.5%), 2 ms (0.8%) and 12 ms (0.7%); and a single burst state with a mean duration of 120 ms. After rundown: mean open time 2.2 ms; five apparent closed states with mean values 0.3 ms (88.9%), 3 ms (3.3%), 19 ms (4.3%), 82 ms (3.1%) and 650 ms (0.4%); and three apparent burst states with mean durations of 23 ms (66%), 5 ms (20.5%) and 0.5 ms (13.5%). Vertical lines indicate the mean values for open times, short closed times and the major burst duration component, and illustrate that rundown has no effect on the intraburst closed times, but reduces the mean open time and burst duration. The methods and solutions used are as described in [6].Download figureOpen in new tabDownload PowerPoint Previous Article VIEW FULL TEXT DOWNLOAD PDF FiguresRelatedReferencesDetailsRelated articlesRunning out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels05 August 2016Philosophical Transactions of the Royal Society B: Biological Sciences This Issue05 September 2016Volume 371Issue 1702Theme issue ‘From DNA barcodes to biomes’ compiled and edited by Paul D. N. Hebert, Mehrdad Hajibabaei and Peter M. Hollingsworth Article InformationDOI:https://doi.org/10.1098/rstb.2016.0330PubMed:27481794Published by:Royal SocietyPrint ISSN:0962-8436Online ISSN:1471-2970History: Published online05/09/2016Published in print05/09/2016 License:© 2016 The Authors.Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Citations and impact Subjectsbiophysicsphysiology

An allosteric model of the inositol trisphosphate receptor with nonequilibrium binding

The inositol trisphosphate receptor (IPR) is a crucial ion channel that regulates the Ca2+ influx from the endoplasmic reticulum (ER) to the cytoplasm. A thorough study of the IPR channel contributes to a better understanding of calcium oscillations and waves. It has long been observed that the IPR channel is a typical biological system which performs adaptation. However, recent advances on the physical essence of adaptation show that adaptation systems with a negative feedback mechanism, such as the IPR channel, must break detailed balance and always operate out of equilibrium with energy dissipation. Almost all previous IPR models are equilibrium models assuming detailed balance and thus violate the dissipative nature of adaptation. In this article, we constructed a nonequilibrium allosteric model of single IPR channels based on the patch-clamp experimental data obtained from the IPR in the outer membranes of isolated nuclei of the Xenopus oocyte. It turns out that our model reproduces the patch-clamp experimental data reasonably well and produces both the correct steady-state and dynamic properties of the channel. Particularly, our model successfully describes the complicated bimodal [Ca2+] dependence of the mean open duration at high [IP3], a steady-state behavior which fails to be correctly described in previous IPR models. Finally, we used the patch-clamp experimental data to validate that the IPR channel indeed breaks detailed balance and thus is a nonequilibrium system which consumes energy.

Baseline Properties of Slo1 K+ (BK) Channels Without the Gating Ring

High conductance Ca2+- and voltage-activated K+ (Slo1 or BK) channels have two functional domains: a “core” consisting of four voltage sensors controlling an ion-conducting pore, and a larger “tail” that forms an intracellular gating ring thought to confer Ca2+ and Mg2+ sensitivity. Whereas the modular structure is known, the properties of the individual modules and the transduction pathways among the modules are poorly understood because it has not been possible to study the core in isolation. To approach this problem, we developed novel constructs that allow functional cores of Slo1 channels to be expressed in the absence of the gating ring. One of these constructs replaced the 827 amino acid gating ring with a short synthetic peptide of 11 amino acids to enhance synthesis and surface expression. Functional expression of this construct in Xenopus oocytes produced currents from channels that: displayed voltage activation with a right-shifted G-V relation; lacked Ca2+ and Mg2+ sensitivity; exhibited a six-fold reduction in mean open interval and burst duration; had an apparent ∼30% reduction in single-channel conductance; and were blocked by extracellular iberiotoxin and TEA and displayed slowed kinetics when co-expressed with β1 subunits. The extent to which any altered channel properties when compared to WT channels reflect the absence of allosteric input from the gating ring, revealing the baseline properties of the core, or reflect input from the 11 amino acid replacement tail is under investigation. Supported in part by NS061871 to LS and AR032805 to KLM.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

Cytoplasmic [InsP3] Drop Induces Transient High-Open-Probability Gating Mode in Type 1 InsP3R Channel

Single-channel activities of homotetrameric recombinant rat type 1 InsP3R channels expressed in mutant DT40-3KO cells with no endogenous InsP3R expression were studied in nuclear patch clamp experiments. In on-nucleus experiments with steady ligand conditions, the channel exhibits modal gating with at least two modes: a high-Po mode with Po = 0.4-0.6 and mean open duration (to) ∼ 3 ms; and a low-Po mode with Po < 0.05 but to still ∼ 3 ms. Cytoplasmic free Ca2+ concentration ([Ca2+]i) regulates channel Po biphasically by changing the likelihood of the channel being in the high-Po mode and the mean closed duration (tc) of the low-Po mode. Even under optimal ligand conditions ([Ca2+]i = 2 µM and cytoplasmic InsP3 concentration [InsP3] = 10 µM), the maximum mean Po observed was only ∼ 0.1, with the channel being in the low-Po mode the majority of time. InsP3 (33 nM to 10 µM) activates the channel by reducing its sensitivity to Ca2+ inhibition. In experiments with cytoplasmic-side-out nuclear membrane patches, when initial [Ca2+]i was low (55 nM) and [InsP3] was rapidly dropped from 10 µM to 0 using perfusion solution exchanges, the InsP3R channels had a high propensity to transiently enter a more active state before they became inactivated by the removal of InsP3. This behavior was more prominent, with the channel Po transiently increasing to ∼ 0.6, when [Ca2+]i was increased to optimal level (2.1 µM) instead of remaining constant at 55 nM. No transient increase in Po was observed in similar [InsP3] drops with [Ca2+]i kept constant at 2.1 µM. This suggests that sub-saturating [InsP3] can induce type 1 InsP3R channels to enter the high-Po mode with higher probability than saturating [InsP3].

TARP‐associated AMPA receptors display an increased maximum channel conductance and multiple kinetically distinct open states

Fast excitatory synaptic transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs), whose biophysical properties are dramatically modulated by the presence of transmembrane AMPAR regulatory proteins (TARPs). To help construct a kinetic model that will realistically describe native AMPAR/TARP function, we have examined the single-channel properties of homomeric GluA1 AMPARs in combination with the TARPs, γ-2, γ-4 and γ-5. In a saturating concentration of agonist, each of these AMPAR/TARP combinations gave rise to single-channel currents with multiple conductance levels that appeared intrinsic to the receptor-channel complex, and showed long-lived subconductance states. The open time and burst length distributions of the receptor complexes displayed multiple dwell-time components. In the case of γ-2- and γ-4-associated receptors, these distributions included a long-lived component lasting tens of milliseconds that was absent from both GluA1 alone and γ-5-associated receptors. The open time distributions for each conductance level required two dwell-time components, indicating that at each conductance level the channel occupies a minimum of two kinetically distinct open states. We have explored how these data place novel constraints on possible kinetic models of TARP-associated AMPARs that may be used to define AMPAR-mediated synaptic transmission.

The GABRA6 mutation, R46W, associated with childhood absence epilepsy, alters α6β2γ2 and α6β2δ GABAA receptor channel gating and expression

A GABA(A) receptor α6 subunit mutation, R46W, was identified as a susceptibility gene that may contribute to the pathogenesis of childhood absence epilepsy (CAE), but the molecular basis for alteration of GABA(A) receptor function is unclear. The R46W mutation is located in a region homologous to a GABA(A) receptor γ2 subunit missense mutation, R82Q, that is associated with CAE and febrile seizures in humans. To determine how this mutation reduces GABAergic inhibition, we expressed wild-type (α6β2γ2L and α6β2δ) and mutant (α6(R46W)β2γ2L and α6(R46W)β2δ) receptors in HEK 293T cells and characterize their whole-cell and single-channel currents, and surface and total levels. We demonstrated that gating and assembly of both α6(R46W)β2γ2L and α6(R46W)β2δ receptors were impaired. Compared to wild-type currents, α6(R46W)β2γ2L and α6(R46W)β2δ receptors had a reduced current density, α6(R46W)β2γ2L currents desensitized to a greater extent and deactivated at a slower rate, α6(R46W)β2δ receptors did not desensitize but deactivated faster and both α6(R46W)β2γ2L and α6(R46W)β2δ single-channel current mean open times and burst durations were reduced. Surface levels of coexpressed α6(R46W), β2 and δ, but not γ2L, subunits were decreased. 'Heterozygous' coexpression of α6(R46W) and α6 subunits with β2 and γ2L subunits produced intermediate macroscopic current amplitudes by increasing incorporation of wild-type and decreasing incorporation of mutant subunits into receptors trafficked to the surface. Finally, these findings suggest that similar to the γ2(R82Q) mutation, the CAE-associated α6(R46W) mutation could cause neuronal disinhibition and thus increase susceptibility to generalized seizures through a reduction of αβγ and αβδ receptor function and expression.

Mg2+ binding to open and closed states can activate BK channels provided that the voltage sensors are elevated

BK channels are activated by intracellular Ca2+ and Mg2+ as well as by depolarization. Such activation is possible because each of the four subunits has two high-affinity Ca2+ sites, one low-affinity Mg2+ site, and a voltage sensor. This study further investigates the mechanism of Mg2+ activation by using single-channel recording to determine separately the action of Mg2+ on the open and closed states of the channel. To limit Mg2+ action to the Mg2+ sites, the two high-affinity Ca2+ sites are disabled by mutation. When the voltage is stepped from negative holding potentials to +100 mV, we find that 10 mM Mg2+ decreases the mean closed latency to the first channel opening 2.1-fold, decreases the mean closed interval duration 8.7-fold, increases mean burst duration 10.1-fold, increases the number of openings per burst 4.4-fold, and increases mean open interval duration 2.3-fold. Hence, Mg2+ can bind to closed BK channels, increasing their opening rates, and to open BK channels, decreasing their closing rates. To explore the relationship between Mg2+ action and voltage sensor activation, we record single-channel activity in macropatches containing hundreds of channels. Open probability (Po) is dramatically increased by 10 mM Mg2+ when voltage sensors are activated with either depolarization or the mutation R210C. The increased Po arises from large decreases in mean closed interval durations and moderate increases in mean open interval durations. In contrast, 10 mM Mg2+ has no detectable effects on Po or interval durations when voltage sensors are deactivated with very negative potentials or the mutation R167E. These observations are consistent with a model in which Mg2+ can bind to and alter the gating of both closed and open states to increase Po, provided that one or more voltage sensors are activated.

An acetylcholine receptor lacking both γ and ε subunits mediates transmission in zebrafish slow muscle synapses

Fast and slow skeletal muscle types in larval zebrafish can be distinguished by a fivefold difference in the time course of their synaptic decay. Single-channel recordings indicate that this difference is conferred through kinetically distinct nicotinic acetylcholine receptor (AChR) isoforms. The underlying basis for this distinction was explored by cloning zebrafish muscle AChR subunit cDNAs and expressing them in Xenopus laevis oocytes. Measurements of single-channel conductance and mean open burst duration assigned α2βδε to fast muscle synaptic current. Contrary to expectations, receptors composed of only αβδ subunits (presumed to be α2βδ2 receptors) recapitulated the kinetics and conductance of slow muscle single-channel currents. Additional evidence in support of γ/ε-less receptors as mediators of slow muscle synapses was reflected in the inward current rectification of heterologously expressed α2βδ2 receptors, a property normally associated with neuronal-type nicotinic receptors. Similar rectification was reflected in both single-channel and synaptic currents in slow muscle, distinguishing them from fast muscle. The final evidence for α2βδ2 receptors in slow muscle was provided by our ability to convert fast muscle synaptic currents to those of slow muscle by knocking down ε subunit expression in vivo. Thus, for the first time, muscle synaptic function can be ascribed to a receptor isoform that is composed of only three different subunits. The unique functional features offered by the α2βδ2 receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.

Voltage Sensor Deactivation Inhibits BK Channel Opening by Mg 2+

Functional and structural studies suggest that intracellular Mg2+ activates BK channels through interaction with the voltage-sensing domain (Yang et al. 2007, 2008; Horrigan & Ma 2008; Yuan et al. 2010; Wu et al. 2010). To further explore the mechanism of activation of BK channels by Mg2+ through the low affinity E374/E399 Mg2+ sites located beneath the voltage sensors, we use single-channel analysis to study BK channels mutated to remove the high affinity Ca2+ sites. We find that 10 mM Mg2+ shortens the latency to first channel opening after a voltage jump to +100 mV from −100 mV, consistent with the hypothesis that Mg2+ can bind to the closed channel and shorten the latency. However, it is not clear whether the closed-channel binding occurs when the voltage sensors are deactivated (down) or activated (up). We therefore recorded single-channel activity in macro-patches held at constant −50 mV, where voltage sensors occasionally activate. Under this condition, 10 mM Mg2+ decreases mean closed duration and increases mean open duration. These effects are attenuated at −100 mV, and become negligible at −150 mV, where the voltage sensors are mainly deactivated. For BK channels modified to have deactivated voltage-sensors (R167E), 10 mM Mg2+ has little effect on mean closed and open durations at −50 mV. In contrast, for BK channels modified to have constitutively activated voltage sensors (R210C), 10 mM Mg2+ shortens the mean closed durations and lengthens the mean open durations at −200 mV. The above observations are consistent with a model in which voltage sensor deactivation inhibits BK channel opening by Mg2+. Supported by NIH grant AR32805 and AHA 10POST4490012.

Constraining Gating Actions of Specific Subunits During NMDA Receptor Activation

NMDA receptor activation gating initiates at its extracellular ligand-binding domain (LBD) and culminates in opening of the associated ion channel. Kinetically, this gating transition occurs through sequential intermediaries with at minimum three pre-open and two open states. The overall gating transition occurs in a concerted manner only after each subunit has bound its respective ligand. Functional NMDA receptors, however, are obligate heterotetramers, typically composed of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. To study subunit-specific contributions to gating kinetics, we constrained relative movements of the M3/M3-S2 central channel gating axis through intra-subunit disulfide cross-linking of partner positions in the M3-S2 and S2-M4 LBD-ion channel linkers, GluN1(C,C) and GluN2A(C,C). Analysis of single-channel recordings revealed the cross-linked receptors to be inefficient in activation gating, with a significantly lower Po [mean SEM; 0.02 ± 0.005 for GluN1(C,C)-GluN2A vs. 0.61 ± 0.05 for GluN1-GluN2A]. This 30-fold reduction in Po is manifested by both a 6-fold decrease in mean open duration (1.3 ± 0.2 vs. 8.3 ± 0.7 ms) and an 18-fold increase in mean closed duration (94 ± 20 vs. 5.1 ± 0.7 ms). The cross-linked receptors undergo only flickery openings to a short-lived open state (τO1, 0.25 ± 0.05 vs. 0.12 ± 0.01 ms) and virtually do not visit the main long-lived (∼ 8.7 ms) open state. Instead, cross-linked receptor activity is locked into two long-lived pre-open states (τC3, 38 ± 9 vs. 4.2 ± 0.4; τC4, 208 ± 45 vs. 52 ± 3 ms). Thus, GluN1 subunit-specific constraint of its M3/M3-S2 gating axis most strongly affects the kinetic end states of activation gating, with minimal, if any, perturbation of the intermediate states. These results support a tightly coupled LBD to ion channel gating machinery in the NMDA receptor.

A novel Polymorphism Linked to Epilepsy Encoding a Missense Mutation in the Pre-M1 Region of a6 Subunits Alters the Gating, but not Trafficking, of GABAa Receptors

Charged residues in the pre-M1 region of Cys-loop ligand-gated ion channels affect current activation, desensitization and deactivation, suggesting that these interface residues may be involved in coupling agonist binding to channel gating. Mutations that affect function and/or expression of GABAA receptors (GABARs) have been associated with idiopathic generalized epilepsies (IGEs). The Parallel Sequence Profiling of Ion Channels in Epilepsy Project at Baylor College of Medicine funded by NINDS and NHGRI identified a novel single nucleotide polymorphism encoding a missense mutation, Q237R, in the GABAR α6 subunit in a patient with an IGE (http://www.hgsc.bcm.tmc.edu/project-medseq-r-ionchannel.hgsc). Homology modeling suggested that Q237R is located within the β10-M1linker or “pre-M1” segment of the N-terminal α6 subunit extracellular domain where it contributes to GABAR subunit-subunit interfaces. We studied gating properties and surface expression of wild type (wt) α6β2γ2 and mutant α6(Q237R)β2γ2 receptors expressed in HEK293T cells. Transient currents were evoked using ultrafast application of 400-µsec pulses of saturating GABA (1 mM) applied to excised outside-out macropatches. Peak amplitudes of α6(Q237R)β2γ2 currents were not reduced but α6(Q237R)β2γ2 currents had slower activation (∼1.9 msec), slower and more desensitization (∼60 %), and faster deactivation (∼2-fold) than wt currents. On-cell α6(Q237R)β2γ2 and α6β2γ2 single channel currents had similar mean open durations, but opening and burst frequencies were significantly higher for mutant than for wt currents. We assessed surface and total cellular expression of wt and mutant receptors using flow cytometry. Co-expression of α6(Q237R) with β2 and γ2L subunits resulted in no significant reduction in total or surface expression of any subunit. We concluded that this mutation alters gating, but not trafficking, of α6β2γ2 GABARs.

Barbiturates Require the N Terminus and First Transmembrane Domain of the δ Subunit for Enhancement of α1β3δ GABAA Receptor Currents

GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization.

Stationary Gating of GluN1/GluN2B Receptors in Intact Membrane Patches

NMDA receptors are heteromeric glutamate-gated channels composed of GluN1 and GluN2 subunits. Receptor isoforms that differ in their GluN2-subunit type (A–D) are expressed differentially throughout the central nervous system and have distinct kinetic properties in recombinant systems. How specific receptor isoforms contribute to the functions generally attributed to NMDA receptors remains unknown, due in part to the incomplete functional characterization of individual receptor types and unclear molecular composition of native receptors. We examined the stationary gating kinetics of individual rat recombinant GluN1/GluN2B receptors in cell-attached patches of transiently transfected HEK293 cells and used kinetic analyses and modeling to describe the full range of this receptor's gating behaviors. We found that, like GluN1/GluN2A receptors, GluN1/GluN2B receptors have three gating modes that are distinguishable by their mean open durations. However, for GluN1/GluN2B receptors, the modes also differed markedly in their mean closed durations and thus generated a broader range of open probabilities. We also found that regardless of gating mode, glutamate dissociation occurred ∼4-fold more slowly (k− = 15 s−1) compared to that observed in GluN1/GluN2A receptors. On the basis of these results, we suggest that slow glutamate dissociation and modal gating underlie the long heterogeneous activations of GluN1/GluN2B receptors.

Voltage Sensor Activation Facilitates Magnesium-Gated Channel Opening in BK Channels

Under physiological conditions, Mg2+ is an intracellular activator of Ca2+- and voltage-activated potassium (BK) channels. To investigate gating by Mg2+ acting through its low affinity site located under each S4 voltage sensor (Yang et al. 2007, 2008; Horrigan & Ma 2008), we studied a BK channel in which the high affinity Ca2+ sites in both the RCK1 domain and the calcium bowl were disabled by mutation. Using single-channel recording from inside-out patches to measure channel activation after voltage steps from −100 mV to +100 mV, we found that 10 mM Mg2+ reduces the latency to first opening after the voltage step and increases channel activation through an increase in the number of openings per burst and mean open duration. This suggests that Mg2+ can bind to both closed and open states of the channel, but it is not clear whether the closed-state binding occurs at the negative or positive potential. Therefore we recorded single-channel activity in macro-patches held at a constant −50 or −100 mV. At −50 mV, when the voltage sensors are occasionally activated, 10 mM Mg2+ decreases the duration of the closed intervals between bursts of activity and increases burst duration through an increase in the number of openings per burst and mean open duration. At −100 mV, when the voltages sensors are mainly deactivated, 10 mM Mg2+ has little effect on the closed intervals between bursts or mean open duration, and most of the bursts are unitary, consisting of a single brief opening. The above data are consistent with a model in which Mg2+ can bind to the BK channel in the closed conformation when the voltage sensors are activated. The bound Mg2+ then facilitates opening. Supported by NIH grant AR32805.

Control of NMDA Receptor Function by the NR2 Subunit Amino-Terminal Domain

NMDA receptors comprised of different NR2 subunits exhibit strikingly unique biophysical and pharmacological properties. Here, we report that the extracellular amino-terminal domain (ATD) of the NR2 subunit controls pharmacological and kinetic properties of recombinant NMDA receptors, such as agonist potency, deactivation time course, open probability (P(OPEN)), and mean open/shut duration. Using ATD deletion mutants of NR2A, NR2B, NR2C, NR2D, and chimeras of NR2A and NR2D with interchanged ATD [NR2A-(2D-ATD) and NR2D-(2A-ATD)], we show that the ATD contributes to the low glutamate potency of NR2A-containing NMDA receptors and the high glutamate potency of NR2D-containing receptors. The ATD influences the deactivation time courses of NMDA receptors, as removal of the ATD from NR2A slows the deactivation rate, while removal of the ATD from NR2B, NR2C and NR2D accelerates the deactivation rate. Open probability also is influenced by the ATD. Removal of the ATD from NR2A or replacement of the NR2A-ATD with that of NR2D decreases P(OPEN) in single-channel recordings from outside-out patches of HEK 293 cells. In contrast, deletion of the ATD from NR2D or replacement of the NR2D ATD with that of NR2A increases P(OPEN) and mean open duration. These data demonstrate the modular nature of NMDA receptors, and show that the ATD of the different NR2 subunits plays an important role in fine-tuning the functional properties of the individual NMDA receptor subtypes.