scholarly journals The activation mechanism of α1β2γ2S and α3β3γ2S GABAA receptors

2009 ◽  
Vol 135 (1) ◽  
pp. 59-75 ◽  
Author(s):  
Angelo Keramidas ◽  
Neil L. Harrison
Keyword(s):  
Gabaa Receptors ◽  
Single Channel ◽  
Activation Mechanism ◽  
Hek293 Cells ◽  
Wide Range ◽  
Single Channel Activity ◽  
Activation Mechanisms ◽  
Sequential Binding ◽  
Kinetic Effects ◽  
Single Channel Currents

The α1β2γ2 and α3β3γ2 are two isoforms of γ-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. α1β2γ2 channels exhibited briefer active periods than α3β3γ2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at α3β3γ2 channels was ∼2.6 µM compared with ∼19 µM for α1β2γ2 channels, suggesting that GABA binds to the α3β3γ2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the α3β3γ2 channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain α3β3γ2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of GABAA receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate GABAA receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system.

scholarly journals Spontaneous single-channel activity of mouse TRP5 channel recombinantly expressed in HEK293 cells

2000 ◽  
Vol 82 ◽  
pp. 131
Author(s):  
Minoru Wakamori ◽  
Hisanobu Yamada ◽  
Takaharu Okada ◽  
Keiji Imoto ◽  
Yasuo Mori
Keyword(s):  
Single Channel ◽  
Hek293 Cells ◽  
Channel Activity ◽  
Single Channel Activity

scholarly journals Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

1987 ◽  
Vol 90 (3) ◽  
pp. 375-395 ◽  
Author(s):  
E Recio-Pinto ◽  
D S Duch ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Planar Bilayers ◽  
Electric Eel ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Single Channel Currents ◽  
Channel Currents

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

scholarly journals Gating reaction mechanism of neuronal NMDA receptors

2012 ◽  
Vol 108 (11) ◽  
pp. 3105-3115 ◽  
Author(s):  
William F. Borschel ◽  
Jason M. Myers ◽  
Eileen M. Kasperek ◽  
Thomas P. Smith ◽  
Nicholas M. Graziane ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Single Channel ◽  
Receptor Expression ◽  
Activation Mechanism ◽  
Neuronal Cultures ◽  
Kinetic Properties ◽  
Recombinant Receptors ◽  
Sufficient Detail ◽  
Activation Mechanisms

The activation mechanisms of recombinant N-methyl-d-aspartate receptors (NRs) have been established in sufficient detail to account for their single channel and macroscopic responses; however, the reaction mechanism of native NRs remains uncertain due to indetermination of the isoforms expressed and possible neuron-specific factors. To delineate the activation mechanism of native NRs, we examined the kinetic properties of currents generated by individual channels located at the soma of cultured rat neurons. Cells were dissociated from the embryonic cerebral cortex or hippocampus, and on-cell single channel recordings were done between 4 and 50 days in vitro (DIV). We observed two types of kinetics that correlated with the age of the culture. When we segregated recordings by culture age, we found that receptors recorded from early (4–33 DIV) and late (25–50 DIV) cultures had smaller unitary conductances but had kinetic profiles that matched closely those of recombinant 2B- or 2A-containing receptors, respectively. In addition, we examined the effects of cotransfection with postsynaptic density protein 95 or neuropilin tolloid-like protein 1 on recombinant receptors expressed in human embryonic kidney-293 cells. Our results add support to the view that neuronal cultures recapitulate the developmental patterns of receptor expression observed in the intact animal and demonstrate that the activation mechanism of somatic neuronal NRs is similar to that described for recombinant receptors of defined subunit composition.

scholarly journals Voltage- and cold-dependent gating of single TRPM8 ion channels

2011 ◽  
Vol 137 (2) ◽  
pp. 173-195 ◽  
Author(s):  
José A. Fernández ◽  
Roman Skryma ◽  
Gabriel Bidaux ◽  
Karl L. Magleby ◽  
C. Norman Scholfield ◽  
...  
Keyword(s):  
Trp Channels ◽  
Single Channel ◽  
Membrane Depolarization ◽  
State Model ◽  
Hek293 Cells ◽  
General Mechanism ◽  
Channel Kinetics ◽  
Open Probability ◽  
Starting Point ◽  
Wide Range

Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30°C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (Po) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of Po, the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation.

scholarly journals Probing the activation sequence of NMDA receptors with lurcher mutations

2012 ◽  
Vol 140 (3) ◽  
pp. 267-277 ◽  
Author(s):  
Swetha E. Murthy ◽  
Tamer Shogan ◽  
Jessica C. Page ◽  
Eileen M. Kasperek ◽  
Gabriela K. Popescu
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Single Channel ◽  
Receptor Activation ◽  
Dissociation Rate ◽  
Activation Mechanism ◽  
Structural Determinants ◽  
Ionic Flux ◽  
Single Channel Currents ◽  
Nmda Receptor Activation

N-methyl-d-aspartate (NMDA) receptor activation involves a dynamic series of structural rearrangements initiated by glutamate binding to glycine-loaded receptors and culminates with the clearing of the permeation pathway, which allows ionic flux. Along this sequence, three rate-limiting transitions can be quantified with kinetic analyses of single-channel currents, even though the structural determinants of these critical steps are unknown. In inactive receptors, the major permeation barrier resides at the intersection of four M3 transmembrane helices, two from each GluN1 and GluN2 subunits, at the level of the invariant SYTANLAAF sequence, known as the lurcher motif. Because the A7 but not A8 residues in this region display agonist-dependent accessibility to extracellular solutes, they were hypothesized to form the glutamate-sensitive gate. We tested this premise by examining the reaction mechanisms of receptors with substitutions in the lurcher motifs of GluN1 or GluN2A subunits. We found that, consistent with their locations relative to the proposed activation gate, A8Y decreased open-state stability, whereas A7Y dramatically stabilized open states, primarily by preventing gate closure; the equilibrium distribution of A7Y receptors was strongly shifted toward active states and resulted in slower microscopic association and dissociation rate constants for glutamate. In addition, for both A8- and A7-substituted receptors, we noticed patterns of kinetic changes that were specific to GluN1 or GluN2 locations. This may be a first indication that the sequence of discernible kinetic transitions during NMDA receptor activation may reflect subunit-dependent movements of M3 helices. Testing this hypothesis may afford insight into the activation mechanism of NMDA receptors.

scholarly journals γ-Amino Butyric Acid Type A Receptor Mutations at β2N265 Alter Etomidate Efficacy While Preserving Basal and Agonist-dependent Activity

2009 ◽  
Vol 111 (4) ◽  
pp. 774-784 ◽  
Author(s):  
Rooma Desai ◽  
Dirk Ruesch ◽  
Stuart A. Forman
Keyword(s):  
Binding Affinity ◽  
Gabaa Receptors ◽  
Type A ◽  
Activation Mechanism ◽  
Hek293 Cells ◽  
Channel Activity ◽  
Channel Activation ◽  
Direct Activation ◽  
Acid Type ◽  
The Impact

Background Etomidate acts at gamma-Aminobutyric acid type A (GABAA) receptors containing beta2 or beta3, but not beta1 subunits. Mutations at beta residue 265 (Ser in beta1; Asn in beta2 or beta3) profoundly affect etomidate sensitivity. Whether these mutations alter etomidate binding remains uncertain. Methods Heterologously expressed alpha1beta2gamma2L GABAA receptors and receptors with beta2(N265S) or beta2(N265M) mutations were studied electrophysiologically in both Xenopus oocytes and HEK293 cells. Experiments quantified the impact of beta2N265 mutations or substituting beta1 for beta2 on basal channel activation, GABA EC50, maximal GABA efficacy, etomidate-induced leftward shift in GABA responses, etomidate direct activation, and rapid macrocurrent kinetics. Results were analyzed in the context of an established allosteric co-agonist mechanism. Results Mutations produced only small changes in basal channel activity, GABA EC50, maximal GABA efficacy, and macrocurrent kinetics. Relative to wild-type, beta2(N265S) reduced etomidate enhancement of apparent GABA affinity six-fold, and it reduced etomidate direct activation efficacy 14-fold. beta2(N265M) totally eliminated both etomidate modulation of GABA responses and direct channel activation. Mechanism-based analysis showed that the function of both mutants remains consistent with the allosteric co-agonist model and that beta2(N265S) reduced etomidate allosteric efficacy five-fold, whereas etomidate-binding affinity dropped threefold. Experiments swapping beta2 subunits for beta1 indicated that etomidate efficacy is reduced 34-fold, whereas binding affinity drops less than two-fold. Conclusions Mutations at beta2N265 profoundly alter etomidate sensitivity with only small changes in basal and GABA-dependent channel activity. Mutations at the beta2N265 residue or replacement of beta2 with beta1 influence etomidate efficacy much more than binding to inactive receptors.

Spontaneous single-channel activity of neuronal TRP5 channel recombinantly expressed in HEK293 cells

2000 ◽  
Vol 285 (2) ◽  
pp. 111-114 ◽  
Author(s):  
Hisanobu Yamada ◽  
Minoru Wakamori ◽  
Yuji Hara ◽  
Yasuo Takahashi ◽  
Kazuhiko Konishi ◽  
...  
Keyword(s):  
Single Channel ◽  
Hek293 Cells ◽  
Channel Activity ◽  
Single Channel Activity

Long-lasting openings of single slow (L-type) Ca2+ channels in chick embryonic heart cells

1990 ◽  
Vol 259 (2) ◽  
pp. H639-H642 ◽  
Author(s):  
N. Tohse ◽  
N. Sperelakis
Keyword(s):  
Single Channel ◽  
External Solution ◽  
Bay K 8644 ◽  
Kinetic Properties ◽  
Heart Cells ◽  
Embryonic Chick ◽  
Single Channel Activity ◽  
Chick Heart ◽  
Single Channel Currents ◽  
Ca2 Channels

Single-channel currents were recorded in cultured embryonic chick (3-day-old) cardiomyocytes in cell-attached patch-clamp experiments. The patch electrode contained 50 mM Ba2+. The cell was bathed in an external solution containing 150 mM K+ (pH 7.4) at room temperature. Depolarizing pulses above -30 mV, from a holding potential of -80 mV, elicited inward unitary currents. The conductance of the channel for this unitary current was 26 pS. The activity of these channels was completely blocked by nifedipine (3 microM). These results indicate that the channel is a slow (L-type) Ca2+ channel. The channels exhibited long-lasting openings, in addition to conventional brief openings. These long openings resembled the long openings produced by the dihydropyridine Ca2+ agonist BAY K 8644 and resultant mode 2 behavior (Hess et al., Nature Lond. 311: 538-544, 1984). The long-lasting openings were observed in 25 patches out of a total of 29 patches in which single-channel activity was present. High open-state probability (Po) sweeps (with Po greater than 0.65), which mainly contain long-lasting openings, accounted for 20.7% of all sweeps. The open-time histogram for the Ca2+ channels was fitted by two exponential components. The time constants of the two components were 0.45 ms (fast) and 6.30 ms (slow). These kinetic properties were similar to those of the previous reports using BAY K 8644. Thus the slow (L-type) Ca2+ channels in young embryonic chick heart cells naturally produce many long-lasting openings in the absence of any added dihydropyridine Ca2+ agonist.

scholarly journals cAMP Activates TRPC6 Channels via the Phosphatidylinositol 3-Kinase (PI3K)-Protein Kinase B (PKB)-Mitogen-activated Protein Kinase Kinase (MEK)-ERK1/2 Signaling Pathway

2011 ◽  
Vol 286 (22) ◽  
pp. 19439-19445 ◽  
Author(s):  
Bing Shen ◽  
Hiu-Yee Kwan ◽  
Xin Ma ◽  
Ching-On Wong ◽  
Juan Du ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Single Channel ◽  
Mesangial Cells ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Activation Mechanism ◽  
Hek293 Cells ◽  
Glomerular Mesangial Cells ◽  
Whole Cell

cAMP is an important second messenger that executes diverse physiological function in living cells. In this study, we investigated the effect of cAMP on canonical TRPC6 (transient receptor potential channel 6) channels in TRPC6-expressing HEK293 cells and glomerular mesangial cells. The results showed that 500 μm 8-Br-cAMP, a cell-permeable analog of cAMP, elicited [Ca2+]i increases and stimulated a cation current at the whole-cell level in TRPC6-expressing HEK293 cells. The effect of cAMP diminished in the presence of the PI3K inhibitors wortmannin and LY294002 or the MEK inhibitors PD98059, U0126, and MEK inhibitor I. 8-Br-cAMP also induced phosphorylation of MEK and ERK1/2. Conversion of serine to glycine at an ERK1/2 phosphorylation site (S281G) abolished the cAMP activation of TRPC6 as determined by whole-cell and cell-attached single-channel patch recordings. Experiments based on a panel of pharmacological inhibitors or activators suggested that the cAMP action on TRPC6 was not mediated by PKA, PKG, or EPAC (exchange protein activated by cAMP). Total internal fluorescence reflection microscopy showed that 8-Br-cAMP did not alter the trafficking of TRPC6 to the plasma membrane. We also found that, in glomerular mesangial cells, glucagon-induced [Ca2+]i increases were mediated through the cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 signaling pathway. In summary, this study uncovered a novel TRPC6 activation mechanism in which cAMP activates TRPC6 via the PI3K-PKB-MEK-ERK1/2 signaling pathway.

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