Suppression by extracellular K+ of N-methyl-D-aspartate responses in cultured rat hippocampal neurons

1990 ◽  
Vol 64 (5) ◽  
pp. 1361-1367 ◽  
Author(s):  
S. Ozawa ◽  
M. Iino ◽  
K. Tsuzuki
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Reversal Potential ◽  
External Solution ◽  
Dependent Manner ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Experimental Conditions ◽  
K Concentration ◽  
External K

1. The effects of increasing K+ concentration in Mg2(+)-free extracellular solution on N-methyl-D-aspartate (NMDA)-induced current were studied in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. When the K+ concentration in the external solution was increased by replacement of Na+ with isomolar K+, the amplitude of the NMDA-induced current decreased in a concentration-dependent manner. The effect of K+ was almost saturated at 100 mM, when the NMDA response was reduced to 12% of that in K(+)-free, 150 mM Na+ solution. Increasing the external K+ concentration did not affect either the kainate- or quisqualate-induced current in these experimental conditions. 3. Increase in the external K+ concentration reduced the NMDA-induced current almost equally over the whole range of membrane potential tested (-60-30 mV). The reversal potential of the NMDA-induced current was not significantly shifted by the replacement of Na+ with K+. 4. A rise in the external K+ concentration to 100 mM did not reduce the single-channel conductance of the NMDA channel, whereas it reduced the mean open time to about two-thirds of that in the control external solution. 5. The suppressed activation of the NMDA receptor channel in high-K+ environments may have a functional significance to alleviate entry of toxic Ca2+ into neurons of the CNS in pathological conditions such as hypoxia and ischemia.

scholarly journals Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block.

1991 ◽  
Vol 98 (1) ◽  
pp. 163-181 ◽  
Author(s):  
W B Ferguson
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Competitive Inhibition ◽  
Reversal Potential ◽  
K Channel ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Single Channel Current

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.

Expression of 5-HT3 receptors in PC12 cells treated with NGF and 8-Br-cAMP

1992 ◽  
Vol 67 (4) ◽  
pp. 812-819 ◽  
Author(s):  
K. Furukawa ◽  
N. Akaike ◽  
H. Onodera ◽  
K. Kogure
Keyword(s):  
Pc12 Cells ◽  
Reversal Potential ◽  
Inward Rectification ◽  
Inactivation Kinetics ◽  
Induced Response ◽  
Dependent Manner ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Inward Current

1. To determine the functional development of neurons, we applied nerve growth factor (NGF) or 8-bromo-cyclic-adenosine monophosphate (8-Br-cAMP) to PC12 cells and recorded the 5-hydroxytryptamine (5-HT)-induced response by the use of a patch-clamp technique. 2. Cultured PC12 cells expressed 5-HT-sensitive receptors, which are almost absent in untreated cells, in the continuous presence of NGF or 8-Br-cAMP for a period of 10 days. 3. Activation of the receptors by 5-HT produced a transient inward current. In a K(+)-free solution, the reversal potential (E5-HT) of I5-HT was +10.3 mV, and the current-voltage (I-V) relation showed inward rectification at positive potentials. 4. The permeability ratio for monovalent cations was Na+:Li+:K+:Rb+:Cs+ = 1:1.19:0.89:0.94:0.91, indicating that a 5-HT-induced current is passing through the ligand-gated large cation channel. 5. 2-Methyl-5-HT, a specific 5-HT3 agonist, induced a similar inward current, even though the current amplitude was smaller and the activation and inactivation kinetics were slower than those of 5-HT. 6. ICS-205-930, a specific 5-HT3 antagonist, inhibited the 5-HT-induced current in a concentration-dependent manner with a noncompetitive inhibition profile. Spiperone, a 5-HT1 and 5-HT2 families antagonist, and ketanserine, 5-HT2 family antagonist, did not affect the 5-HT-induced response. 7. The time to peak (tp) as well as fast and slow time constants (tau if and tau is) decreased with increasing 5-HT concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a delayed rectifier potassium current in chicken growth plate chondrocytes

1992 ◽  
Vol 262 (5) ◽  
pp. C1335-C1340 ◽  
Author(s):  
K. B. Walsh ◽  
S. D. Cannon ◽  
R. E. Wuthier
Keyword(s):  
Growth Plate ◽  
Potassium Current ◽  
Reversal Potential ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Growth Plate Chondrocytes ◽  
Patch Clamp Technique ◽  
Delayed Rectifier Potassium Current ◽  
Current Activation ◽  
External K

With the use of the whole cell arrangement of the patch-clamp technique, an outward-directed time-dependent potassium current was identified in cultured chicken growth plate chondrocytes. This delayed rectifier potassium current (IK) activated with a sigmoidal time course during voltage steps to potentials positive to -40 mV. The half-maximal voltage required for current activation was determined to be -8 mV. The reversal potential (Erev) for IK, measured using deactivating tail currents, was -72 mV in the presence of 140 mM internal and 5 mM external [K+] solutions. Changes in external [K+] caused Erev to shift in a manner expected for a potassium-selective channel. In addition, increasing external [K+] from 5 to 50 mM caused the slope conductance of the tail currents to increase twofold. The chondrocyte IK was inhibited by the potassium-channel blocker 4-aminopyridine (4-AP) at concentrations of 0.5-4 mM and by the scorpion venom toxin charybdotoxin (CTX; 10 nM) but was unaffected by 10 mM tetraethylammonium (TEA). Addition of 20 microM ZnCl2 reduced IK in a voltage-dependent manner with the greatest inhibition found to occur at potentials near the threshold for current activation. Reduction of IK by ZnCl2 was accompanied by a slowing in the kinetics of IK activation. On the basis of the gating and pharmacological properties of this current, it is suggested that the chondrocyte channel belongs to a superfamily of K+ channels found in bone and immune system cells. The chondrocyte K+ channel may contribute to the unusually high [K+] found in the extracellular fluid of growth plate cartilage.

ATP-induced inward current in neurons freshly dissociated from the tuberomammillary nucleus

1994 ◽  
Vol 71 (3) ◽  
pp. 868-873 ◽  
Author(s):  
K. Furukawa ◽  
H. Ishibashi ◽  
N. Akaike
Keyword(s):  
Purinergic Receptor ◽  
Reversal Potential ◽  
External Solution ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Induced Current ◽  
Excitatory Effect ◽  
Inward Current ◽  
Tuberomammillary Nucleus

1. Neurons in the tuberomammillary nucleus (TMN), which are considered to be histaminergic, were dissociated and their response to extracellularly applied ATP was investigated in the nystatin-perforated patch recording mode under voltage-clamp condition. 2. ATP induced a sustained inward current that was slowly desensitized at a holding potential of -60 mV. 3. The ATP response increased in a concentration-dependent manner. The half-maximum concentration (EC50) was 44 microM and the Hill coefficient was 1.8. 4. The potency of ATP analogues was in the order of ATP > or = 2-methylthio-ATP >> alpha, beta-methylene ATP > or = ADP. Neither adenosine nor AMP induced any response. The results suggest that the purinergic receptor in TMN neurons is P2y. 5. The current-voltage relationship for the 100 microM ATP showed a significant inward rectification at a potential more positive than -20 mV in an external solution with 150 mM Na+, but a significant rectification current was not observed in an external solution with 150 mM Cs+. The change in the reversal potential of the ATP response (EATP) to a 10-fold change of extracellular Na+ concentration was 56 mV, indicating that the ATP-induced current is highly selective for Na+ over Cl-. 6. The permeability ratio for cations was Na+:Li+:K+:Rb+: Cs+:Ca2+ = 2.16:1.36:1.68:1.54:1:2.55, indicating that the ATP-induced current is passing through the ligand-gated nonselective cation channel. 7. These results suggest that ATP has an excitatory effect on the TMN neurons by opening nonselective cation channels.

Two types of kainate response in cultured rat hippocampal neurons

1991 ◽  
Vol 66 (1) ◽  
pp. 2-11 ◽  
Author(s):  
S. Ozawa ◽  
M. Iino ◽  
K. Tsuzuki
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Pyramidal Cells ◽  
Nmda Receptor Antagonist ◽  
Inward Rectification ◽  
Type I ◽  
Type Ii ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Whole Cell

1. Two different types of kainate response were recorded in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. There was an outward rectification in the current-voltage (I-V) plot of the kainate-induced current (type I response) in relatively large neurons bearing a morphological resemblance to young pyramidal cells. In smaller neurons with elliptical somata and fine neurites, the kainate response was characterized by a remarkable inward rectification in the I-V plot of the kainate-induced current and a significant permeability to Ca2+ (type II response). 3. Both type I and type II responses were negligible below 2 microM and almost saturated at 500 microM kainate. The concentrations producing half-maximal responses and the Hill coefficients were 68 microM and 1.76 and 56 microM and 1.21 for type I and type II responses, respectively. Both responses were suppressed similarly by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 4. The mean single-channel conductance (gamma) of the type II kainate response was estimated, from the relation between the whole-cell mean currents and current variances, to be 8.7 pS. The power spectrum for the current noise was fitted with the sum of two Lorentzians with cutoff frequencies (fc) of 61.1 +/- 1.4 and 327.8 +/- 10.5 Hz (n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

Single voltage-dependent potassium channels in cultured rat hippocampal neurons

1986 ◽  
Vol 56 (2) ◽  
pp. 481-493 ◽  
Author(s):  
M. A. Rogawski
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Reversal Potential ◽  
Resting Potential ◽  
Moderate Degree ◽  
Patch Clamp Technique ◽  
Membrane Pore ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Single Channel Currents

Single-channel recordings using the gigohm seal patch-clamp technique were carried out on the somatic membranes of dissociated embryonic rat hippocampal neurons grown in cell culture. The recording medium contained tetrodotoxin to block the voltage-dependent Na+ conductance and Cd2+ to block Ca2+ and Ca2+-activated conductances. In the cell-attached configuration, depolarizing voltage steps activated outward directed single-channel currents with conductance 15-20 pS. The channel openings exhibited a moderate degree of flickering. The mean burst lifetimes ranged from 5 to 13 ms with a tendency to increase slightly at more depolarized potentials (T = 21-25 degrees C). Reversal potential measurements using excised membrane patches indicated that the channels behaved as expected of a K+-selective membrane pore. Channel opening occurred in Ca2+-free EGTA-containing solutions but was never observed in the presence of tetraethylammonium (TEA; 20 mM). The frequency of channel opening increased as the membrane was depolarized by up to 50 mV from resting potential; the fraction of time spent in the open state during the first 300 ms following a step depolarization increased e-fold for a 8-25 mV change in potential. First-latency histograms and simulations of the macroscopic current based on channel data obtained during repeated depolarizing voltage steps indicated that the probability of the channel being in the open state increases gradually with time after a step depolarization. During repeated depolarizing steps the channels appeared to randomly enter and exit a long-lived inactive state. It is concluded that these channels may underly the slowly activating, very slowly inactivating, TEA-sensitive voltage-dependent K+ current (IK) in cultured hippocampal neurons.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

Single-channel analysis of a K channel at basolateral membrane of rabbit proximal convoluted tubule

1988 ◽  
Vol 254 (1) ◽  
pp. F105-F113 ◽  
Author(s):  
L. Parent ◽  
J. Cardinal ◽  
R. Sauve
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
Time Interval ◽  
Distribution Analysis ◽  
Patch Clamp Technique ◽  
Bathing Medium ◽  
Patch Pipette ◽  
K Concentration ◽  
Interval Distribution

The basolateral membrane of the rabbit proximal convoluted tubule (PCT) is known to be largely permeable to K ions. The patch-clamp technique was used to investigate the molecular basis of this K permeability. At room temperature and with a high-K solution (127 mM) in both the bathing medium and the patch pipette, current jumps associated with an inward-rectifying channel could be detected in every active cell-attached experiment. When the K concentration in the pipette was changed from 200 to 5 mM KCl (NaCl replacement), the single-channel conductance for inward currents changed from 54 to 10 pS. The observed shift in the zero current potential measured as a function of the patch pipette K concentration could be fitted using the Goldman-Hodgkin-Katz equation with a permeability ratio PNa/PK = 0.06. The channel was found to be moderately voltage dependent (e-fold per 56 mV depolarization). For instance, the open-channel probability (Po) increased from 0.06 to 0.16 following a membrane depolarization from -50 to +50 mV. A time interval distribution analysis showed for the open state a dominant single time constant of 14 and 10 ms at 50 and -50 mV, respectively. Two time constants equal to 1 (flickering) and 26 ms at +50 mV and to 0.6 and 300 ms at -50 mV were obtained for the closed-state interval distribution. Based on this analysis, it was concluded that the decrease of Po at negative potentials was due more to the appearance of prolonged silent periods than from a change in the channel mean open time.

Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells

1986 ◽  
Vol 251 (1) ◽  
pp. C85-C89 ◽  
Author(s):  
N. W. Richards ◽  
D. C. Dawson
Keyword(s):  
Epithelial Cells ◽  
Single Channel ◽  
Reversal Potential ◽  
Cell Swelling ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Colon Cells ◽  
High K ◽  
A Cell ◽  
Single Channel Currents

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.

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