Sites of antagonist action on N-methyl-D-aspartic acid receptors studied using fluctuation analysis and a rapid perfusion technique

1988 ◽  
Vol 60 (2) ◽  
pp. 645-663 ◽  
Author(s):  
M. L. Mayer ◽  
G. L. Westbrook ◽  
L. Vyklicky
Keyword(s):  
Nmda Receptor ◽  
Kynurenic Acid ◽  
Single Channel ◽  
Nmda Antagonist ◽  
Fluctuation Analysis ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean

1. Mouse hippocampal neurons in dissociated culture were grown at low density on previously plated hippocampal glial cell cultures and voltage clamped using the tight seal whole-cell patch-clamp technique. Flow pipes were used to rapidly exchange the extracellular solution, and to apply N-methyl-D-aspartic acid (NMDA) and some NMDA antagonists. Fluctuation analysis was used to estimate changes in the behavior of NMDA-activated ion channels during application of antagonists. In the presence of NMDA control spectra were well fit by single Lorentzian functions consistent with mean open times of 5-6 ms. 2. Two antagonists thought to act at the NMDA receptor agonist recognition site, 2-amino-5-phosphonovaleric acid (AP5) and kynurenic acid, did not produce changes in the mean open time or single channel conductance, consistent with their action as competitive antagonists. Onset of antagonism and recovery from the action of both AP5 and kynurenic acid was rapid and complete within 1 s. However, raising the extra-cellular glycine concentration, from 1 microM to 1 mM, reduced the potency of 100 microM kynurenic acid as an NMDA antagonist, suggesting that kynurenate has an additional action as a competitive antagonist at the glycine modulatory site on NMDA receptor channels. 3. In the presence of 150 microM magnesium NMDA spectra recorded at -60 mV were fit by double Lorentzian functions, consistent with single-channel events consisting of bursts of openings lasting 3.3 ms in duration, interrupted by blocking and unblocking events of average duration 0.18 ms. The onset and recovery from magnesium antagonism was rapid, and complete within 1 s, but was highly voltage dependent and at +40 mV magnesium (150 microM) failed to produce NMDA antagonism. These results are consistent with a voltage-dependent channel block of NMDA receptor channels produced by binding of magnesium to a site within the ion channel. 4. Zinc (30 microM) was a potent NMDA antagonist at both -60 and +40 mV, and at either potential appeared to reduce the mean open time of NMDA-activated ion channels from about 5 ms to approximately 3 ms. Over the frequency range measured, 1-1,000 Hz, NMDA spectra were well fit by single Lorentzians during zinc antagonism, in contrast to results obtained with magnesium. The mean single channel conductance also decreased in the presence of zinc to approximately 75% of control. Onset of antagonism and recovery from the action of zinc was rapid and complete within 1 s.(ABSTRACT TRUNCATED AT 400 WORDS)

Basolateral potassium channels in renal proximal tubule

1987 ◽  
Vol 253 (3) ◽  
pp. F476-F487 ◽  
Author(s):  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
K Channels ◽  
Open Time ◽  
Excised Patches ◽  
The Mean ◽  
Inside Out

Potassium (K+) channels in the basolateral membrane of unperfused Necturus proximal tubules were studied in both cell-attached and excised patches, after removal of the tubule basement membrane by manual dissection without collagenase. Two different K+ channels were identified on the basis of their kinetics: a short open-time K+ channel, with a mean open time less than 1 ms, and a long open-time K+ channel with a mean open time greater than 20 ms. The short open-time channel occurred more frequently than the longer channel, especially in excised patches. For inside-out excised patches with Cl- replaced by gluconate, the current-voltage relation of the short open-time K+ channel was linear over +/- 60 mV, with a K+-Na+ selectivity of 12 +/- 2 (n = 12), as calculated from the reversal potential with oppositely directed Na+ and K+ gradients. With K-Ringer in the patch pipette and Na-Ringer in the bath, the conductance of the short open-time channel was 47 +/- 2 pS (n = 15) for cell-attached patches, 26 +/- 2 pS (n = 15) for patches excised (inside out) into Na-Ringer, and 36 +/- 6 pS (n = 3) for excised patches with K-Ringer on both sides. These different conductances can be partially explained by a dependence of single-channel conductance on the K+ concentration on the interior side of the membrane. In experiments with a constant K+ gradient across excised patches, large changes in Na+ at the interior side of the membrane produced no change in single-channel conductance, arguing against a direct block of the K+ channel by Na+. Finally, the activity of the short open-time channel was voltage gated, where the mean number of open channels decreased as a linear function of basolateral membrane depolarization for potentials between -60 and 0 mV. Depolarization from -60 to -40 mV decreased the mean number of open K+ channels by 28 +/- 8% (n = 6).

Chloride efflux inhibits single calcium channel open probability in vertebrate photoreceptors: Chloride imaging and cell-attached patch-clamp recordings

2000 ◽  
Vol 17 (2) ◽  
pp. 197-206 ◽  
Author(s):  
WALLACE B. THORESON ◽  
RON NITZAN ◽  
ROBERT F. MILLER
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Pipette Solution ◽  
Open Time ◽  
The Mean ◽  
Channel Properties ◽  
Distribution Fit

The present study uses cell-attached patch-recording techniques to study the single-channel properties of Ca2+ channels in isolated salamander photoreceptors and investigate their sensitivity to reductions in intracellular Cl−. The results show that photoreceptor Ca2+ channels possess properties similar to L-type Ca2+ channels in other preparations, including (1) enhancement of openings by the dihydropyridine agonist, (−)BayK8644; (2) suppression by a dihydropyridine antagonist, nisoldipine; (3) single-channel conductance of 22 pS with 82 mM Ba2+ as the charge carrier; (4) mean open probability of 0.1; (5) open-time distribution fit with a single exponential (τ0 = 1.1 ms) consistent with a single open state; and (6) closed time distribution fit with two exponentials (τc1 = 0.7 ms, τc2 = 25.4 ms) consistent with at least two closed states. Using a Cl−-sensitive dye to measure intracellular [Cl−], it was found that perfusion with gluconate-containing, low Cl− medium depleted intracellular [Cl−]. It was therefore possible to reduce intracellular [Cl−] by perfusion with a low Cl− solution while maintaining the extracellular channel surface in high Cl− pipette solution. Under these conditions, the single-channel conductance was unchanged, but the mean open probability fell to 0.03. This reduction can account for the 66% reduction in whole-cell Ca2+ currents produced by perfusion with low Cl− solutions. Examination of the open and closed time distributions suggests that the reduction in open probability arises from increases in closed-state dwell times. Changes in intracellular [Cl−] may thus modulate photoreceptor Ca2+ channels.

scholarly journals A voltage-clamp study of isolated stingray horizontal cell non-NMDA excitatory amino acid receptors

1989 ◽  
Vol 61 (1) ◽  
pp. 162-172 ◽  
Author(s):  
T. J. O'Dell ◽  
B. N. Christensen
Keyword(s):  
Amino Acid ◽  
Dicarboxylic Acid ◽  
Single Channel ◽  
Excitatory Amino Acid ◽  
Rank Order ◽  
Horizontal Cell ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Time ◽  
The Mean

1. Horizontal cells enzymatically isolated from retinas of the Atlantic stingray (Dasyatis sabina) were voltage-clamped using the patch electrode in the whole-cell mode. A rapid microsuperfusion system was used to apply excitatory amino acid agonists and antagonists. 2. The isolated cells responded to glutamate (GLU), kainate (KA), quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Responses elicited by GLU, QA, and AMPA but not KA exhibited a concentration-dependent and concanavalin A- (Con-A) sensitive desensitization. No responses were elicited by aspartate, N-methyl-D-aspartate, or quinolinate at concentrations as high as 1.0 mM. 3. Judging from the concentration producing one-half of the maximal current response (EC50), the rank order affinities of the agonists was QA greater than or equal to GLU greater than AMPA greater than KA. Whereas KA had the lowest affinity of the agonists tested it was the most efficacious, producing the largest currents. Hill coefficients of the concentration-response data were near two for KA and GLU and near one for QA and AMPA. 4. The agonists differed in their sensitivity to various excitatory amino acid receptor antagonists. Kynurenate (KYN) produced a nearly complete block of horizontal cell responses to GLU and KA at concentrations that had little effect on QA and AMPA. Piperidine-2,3-dicarboxylic acid (cis-PDA), 1-(4-chlorobenzoyl)-piperazine-2,3-dicarboxylic acid (pCB-PzDA), and folic acid were less potent antagonists than KYN but were also better blockers of KA and GLU responses than of QA- and AMPA-elicited responses. 5. When QA, AMPA, or GLU were applied in combination with 55.0 microM KA the current was less than that produced by KA alone. The rank order potency for the inhibition of KA-elicited responses was QA greater than AMPA greater than GLU. In the presence of low concentrations of KA (1.0-20.0 microM), QA- and AMPA-elicited responses were potentiated. This potentiation was prevented by KYN. 6. Single-channel conductance and mean open time were estimated from the current noise fluctuations in the presence of agonist. The mean single-channel conductance for QA was 9 pS that was almost twice as large as the conductance for KA (5.9 pS) and GLU (5.7 pS). The mean open time in the presence of QA or GLU was approximately 1 ms, which was about one-half of that for KA (2.0 ms). 7. These results are best explained by the existence of a single receptor protein with multiple but not identical ligand-binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of NMDA Receptors in Rat Dentate Gyrus Granule Cells by Spontaneous and Evoked Transmitter Release

2003 ◽  
Vol 90 (2) ◽  
pp. 786-797 ◽  
Author(s):  
Nils Ole Dalby ◽  
Istvan Mody
Keyword(s):  
Nmda Receptor ◽  
Dentate Gyrus ◽  
Nmda Receptors ◽  
Granule Cells ◽  
Single Channel ◽  
Complex Mixture ◽  
Fluctuation Analysis ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Current Shift

Activation of N-methyl-d-aspartate (NMDA) receptors by synaptically released glutamate in the nervous system is usually studied using evoked events mediated by a complex mixture of AMPA, kainate, and NMDA receptors. Here we have characterized pharmacologically isolated spontaneous NMDA receptor-mediated synaptic events and compared them to stimulus evoked excitatory postsynaptic currents (EPSCs) in the same cell to distinguish between various modes of activation of NMDA receptors. Spontaneous NMDA receptor-mediated EPSCs recorded at 34°C in dentate gyrus granule cells (DGGC) have a frequency of 2.5 ± 0.3 Hz and an average peak amplitude of 13.2 ± 0.8 pA, a 10–90% rise time of 5.4 ± 0.3 ms, and a decay time constant of 42.1 ± 2.1 ms. The single-channel conductance estimated by nonstationary fluctuation analysis was 60 ± 5 pS. The amplitudes (46.5 ± 6.4 pA) and 10–90% rise times (18 ± 2.3 ms) of EPSCs evoked from the entorhinal cortex/subiculum border are significantly larger than the same parameters for spontaneous events (paired t-test, P < 0.05, n = 17). Perfusion of 50 μM d(–)-2-amino-5-phosphonopentanoic acid blocked all spontaneous activity and caused a significant baseline current shift of 18.8 ± 3.0 pA, thus identifying a tonic conductance mediated by NMDA receptors. The NR2B antagonist ifenprodil (10 μM) significantly reduced the frequency of spontaneous events but had no effect on their kinetics or on the baseline current or variance. At the same time, the peak current and charge of stimulus-evoked events were significantly diminished by ifenprodil. Thus spontaneous NMDA receptor-mediated events in DGGC are predominantly mediated by NR2A or possibly NR2A/NR2B receptors while the activation of NR2B receptors reduces the excitability of entorhinal afferents either directly or through an effect on the entorhinal cells.

Horizontal cells isolated from catfish retina contain two types of excitatory amino acid receptors

1989 ◽  
Vol 61 (6) ◽  
pp. 1097-1109 ◽  
Author(s):  
T. J. O'Dell ◽  
B. N. Christensen
Keyword(s):  
Nmda Receptor ◽  
Single Channel ◽  
Rank Order ◽  
Horizontal Cells ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Response Curves ◽  
Receptor Agonists ◽  
Open Time ◽  
Hill Coefficients

1. Inward going membrane currents elicited by N-methyl-D-aspartate (NMDA), glutamate (GLU), and glutamate analogues were recorded from isolated catfish (Ictalurus punctatus) cone horizontal cells using the patch-clamp technique in the whole-cell mode. 2. Currents elicited by the N-methyl-D-aspartate receptor agonists NMDA, L-aspartate (ASP) or L-homocysteate (L-HCA) in nominally Mg-free saline were completely blocked by 100 microM 2-amino-5-phosphonovalerate (AP-5) but responses to non-NMDA receptor agonists kainate (KA), quisqualate (QA), or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) in normal Mg2+ saline were unaffected. Responses to GLU were partially blocked. Kynurenic acid (1 mM) effectively blocked responses to all agonists. 3. Concentration-response curves obtained from measured responses in the presence of different NMDA receptor agonists gave Hill coefficients of near 1 indicating a single binding site for channel activation. The rank order of agonist affinity at the NMDA receptor is L-HCA greater than NMDA greater than ASP. Glycine potentiates responses to NMDA in horizontal cells. 4. The NMDA-activated channel is blocked in a voltage-dependent manner by Mg2+, Ni2+, and Co2+ and in a voltage-independent manner by Zn2+. Both the NMDA- and KA-activated channel were permeable to monovalent cations but the NMDA-activated channel appeared to have a greater permeability to Ca2+ than the KA-activated channel. 5. Concentration-response curves measured from responses to the non-NMDA receptor agonists QA, KA, and AMPA gave Hill coefficients of approximately 1.5 suggesting multiple binding sites for channel activation and cooperativity. The rank-order affinity was QA greater than AMPA greater than GLU greater than KA. KA was the most efficacious of the agonists resulting in the largest Imax. Rapid desensitization was observed only in the presence of QA, AMPA, or GLU and this desensitization could be removed by pretreatment with conconavalin A (Con A). 6. Single-channel conductance and mean open time were measured from the fluctuations in current noise in the presence of the agonists. The single-channel conductance estimated from the slope of a linear regression obtained from a plot of the variance of the conductance versus the whole-cell conductance measured in NMDA and ASP was 4.7 pS. The mean channel open time was 1.3 ms. These same parameters measured for KA and QA were 5.7 and 5.9 pS and 1.1 to 1.3 ms, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Gating and Conductance Properties of Bk Channels Are Modulated by the S9–S10 Tail Domain of the α Subunit

2001 ◽  
Vol 118 (6) ◽  
pp. 711-734 ◽  
Author(s):  
Brenda L. Moss ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Burst Duration ◽  
Tail Domain ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Time ◽  
The Mean ◽  
Single Channel Currents

The COOH-terminal S9–S10 tail domain of large conductance Ca2+-activated K+ (BK) channels is a major determinant of Ca2+ sensitivity (Schreiber, M., A. Wei, A. Yuan, J. Gaut, M. Saito, and L. Salkoff. 1999. Nat. Neurosci. 2:416–421). To investigate whether the tail domain also modulates Ca2+-independent properties of BK channels, we explored the functional differences between the BK channel mSlo1 and another member of the Slo family, mSlo3 (Schreiber, M., A. Yuan, and L. Salkoff. 1998. J. Biol. Chem. 273:3509–3516). Compared with mSlo1 channels, mSlo3 channels showed little Ca2+ sensitivity, and the mean open time, burst duration, gaps between bursts, and single-channel conductance of mSlo3 channels were only 32, 22, 41, and 37% of that for mSlo1 channels, respectively. To examine which channel properties arise from the tail domain, we coexpressed the core of mSlo1 with either the tail domain of mSlo1 or the tail domain of mSlo3 channels, and studied the single-channel currents. Replacing the mSlo1 tail with the mSlo3 tail resulted in the following: increased open probability in the absence of Ca2+; reduced the Ca2+ sensitivity greatly by allowing only partial activation by Ca2+ and by reducing the Hill coefficient for Ca2+ activation; decreased the voltage dependence ∼28%; decreased the mean open time two- to threefold; decreased the mean burst duration three- to ninefold; decreased the single-channel conductance ∼14%; decreased the Kd for block by TEAi ∼30%; did not change the minimal numbers of three to four open and five to seven closed states entered during gating; and did not change the major features of the dependency between adjacent interval durations. These observations support a modular construction of the BK channel in which the tail domain modulates the gating kinetics and conductance properties of the voltage-dependent core domain, in addition to determining most of the high affinity Ca2+ sensitivity.

scholarly journals Single calcium channel behavior in native skeletal muscle.

1995 ◽  
Vol 105 (2) ◽  
pp. 227-247 ◽  
Author(s):  
R T Dirksen ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Channels ◽  
Single Channel ◽  
Bay K 8644 ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Whole Cell ◽  
Macroscopic Properties ◽  
Voltage Dependent

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.

scholarly journals Gating of Acid-sensitive Ion Channel-1: Release of Ca2+ Block vs. Allosteric Mechanism

2006 ◽  
Vol 127 (2) ◽  
pp. 109-117 ◽  
Author(s):  
Ping Zhang ◽  
Fred J. Sigworth ◽  
Cecilia M. Canessa
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Single Channel ◽  
Allosteric Mechanism ◽  
Open Time ◽  
Channel Opening ◽  
Voltage Dependent ◽  
High Affinity Binding Site ◽  
The Mean ◽  
High Concentrations

The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at −60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.

Properties of single Na+ channels in cut-open Myxicola giant axons

1987 ◽  
Vol 65 (4) ◽  
pp. 568-573 ◽  
Author(s):  
C. L. Schauf
Keyword(s):  
Patch Clamp ◽  
Time Dependence ◽  
Voltage Dependence ◽  
Single Channel ◽  
Single Channels ◽  
Channel Conductance ◽  
Dependent Behavior ◽  
Open Time ◽  
Voltage Dependent ◽  
Steady State Inactivation

Time- and voltage-dependent behavior of the Na+ conductance in dialyzed intact Myxicola axons was compared with cut-open axons subjected to loose-patch clamp of the interior and to axons where Gigaseals were formed after brief enzyme digestion. Voltage and time dependence of activation, inactivation, and reactivation were identical in whole-axons and loose-patch preparations. Single channels observed in patch-clamp axons had a conductance of 18.3 ± 2.3 pS and a mean open time of 0.84 ± 0.12 ms. The time-dependence of Na+ currents found by averaging patch-clamp records was similar to intact axons, as was the voltage dependence of activation. Steady-state inactivation in patch-clamped axons was shifted by an average of 15 mV from that seen in loose-patch or intact axons. Substitution of D2O for H2O decreased single channel conductance by 24 ± 6% in patch-clamped axons compared with 28 ± 4% in intact axons, slowed inactivation by 58 ± 8% compared with 49 ± 6%, and increased mean open time by 52 ± 7%. The results confirm observations on macroscopic channel behavior in Myxicola and resemble that seen in other excitable tissues.

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