scholarly journals Evidence of Decreased Activity in Intermediate-Conductance Calcium-Activated Potassium Channels During Retinoic Acid–Induced Differentiation in Motor Neuron–Like NSC-34 Cells

2018 ◽  
Vol 48 (6) ◽  
pp. 2374-2388 ◽  
Author(s):  
Pei-Chun Chen ◽  
Jing-Syuna Ruan ◽  
Sheng-Nan Wu
Keyword(s):  
Retinoic Acid ◽  
Motor Neuron ◽  
Messenger Rna ◽  
Single Channel ◽  
Slow Component ◽  
Delayed Rectifier ◽  
Induced Differentiation ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
The Mean

Background/Aims: Intermediate-conductance Ca2+-activated K+ (IKCa; KCa3.1 or KCNN4) channels affect the behaviors of central neurons including motor neurons. The mechanism through which neuronal differentiation is related to the activity of these channels remains largely unclear. Methods: By using various molecular biology tools and electrophysiological measurements, we investigated possible changes in the activity of IKCa channels in a retinoic acid (RA)-induced differentiation process in motor neuron-like NSC-34 cells. Results: The protein and messenger RNA expression of KCa3.1 substantially diminished as NSC-34 cells were differentiated with low serum (1%) and 1 µM RA. In whole-cell current recordings, the density of delayed-rectifier K+ currents obtained from differentiated cells was elevated. However, the density of a ramp pulse-elicited K+ current that was sensitive to blockage by 1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole (TRAM-34)—an inhibitor of IKCa channels—was significantly higher in undifferentiated NSC-34 cells than in differentiated cells. In undifferentiated cells, the activity of IKCa channels was readily detected and the probability of channel openings was resistant to stimulation by diazoxide or suppression by verruculogen. Furthermore, this probability was increased by 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one or 9-phenanthrol and reduced by TRAM-34. The channel-opening probability decreased in RA-induced differentiated cells, whereas the single-channel conductance of IKCa channels did not differ between undifferentiated and differentiated cells. Moreover, the slow component of the mean closed time in these channels was significantly shorter in undifferentiated cells than in differentiated cells; however, the mean open time in the channel remained unchanged as cells were differentiated. Conclusion: RA-induced differentiation in neurons could exert a suppressive effect on the activity of IKCa channels.

scholarly journals Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels

2021 ◽  
Vol 14 (5) ◽  
pp. 388
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Ionic Channel ◽  
Gh3 Cells ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Gating Charge ◽  
Ramp Voltage ◽  
K Current ◽  
The Mean

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

Single HERG delayed rectifier K+ channels expressed in Xenopus oocytes

1997 ◽  
Vol 272 (3) ◽  
pp. H1309-H1314 ◽  
Author(s):  
A. Zou ◽  
M. E. Curran ◽  
M. T. Keating ◽  
M. C. Sanguinetti
Keyword(s):  
Cardiac Myocytes ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
K Channel ◽  
Delayed Rectifier ◽  
Class Iii ◽  
Channel Opening ◽  
The Mean ◽  
K Concentration ◽  
Channel Properties

HERG is a K+ channel with properties similar to the rapidly activating component (I(Kr)) of delayed rectifier K+ current, which is important for repolarization of human cardiac myocytes. In this study, we have characterized the single-channel properties of HERG expressed in Xenopus oocytes. Currents were measured in cell-attached patches with an extracellular K concentration of 120 mM. The single HERG channel conductance, determined at test potentials between -50 and -110 mV, was 12.1 +/- 0.6 pS. At positive test potentials (+40 to +80 mV), the probability of channel opening was low and slope conductance was 5.1 +/- 0.6 pS. The mean channel open times at -90 mV were 2.9 +/- 0.5 and 11.8 +/- 1.0 ms, and the mean channel closed times were 0.54 +/- 0.02 and 14.5 +/- 5.3 ms. Single HERG channels were blocked by MK-499, a class III antiarrhythmic agent that blocks I(Kr) in cardiac myocytes. The development of block was more rapid in inside-out patches than in cell-attached patches or in whole cell recordings, indicating that block occurs from the cytoplasmic side of the membrane. The single-channel properties of HERG are similar to I(Kr) channels of isolated cardiac myocytes, which provides further evidence that HERG proteins coassemble to form I(Kr) channels.

scholarly journals Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions.

1991 ◽  
Vol 98 (1) ◽  
pp. 1-17 ◽  
Author(s):  
E Perozo ◽  
C A Vandenberg ◽  
D S Jong ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Slow Component ◽  
Giant Axon ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Delayed Rectifier Current ◽  
K Current ◽  
The Difference ◽  
Single Channel Currents

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.

Differentiation of NG108-15 cells alters channel conductance and desensitization kinetics of the 5-HT3 receptor

1991 ◽  
Vol 65 (3) ◽  
pp. 630-638 ◽  
Author(s):  
X. M. Shao ◽  
J. L. Yakel ◽  
M. B. Jackson
Keyword(s):  
Single Channel ◽  
Noise Analysis ◽  
Morphological Differentiation ◽  
Culture Conditions ◽  
Receptor Subtypes ◽  
Channel Conductance ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
Current Voltage Curve ◽  
Single Channel Currents

1. NG108-15 cells undergo morphological differentiation in response to appropriate culture conditions. We have used patch clamp techniques to compare responses mediated by the 5-HT3 receptor in differentiated and undifferentiated NG108-15 cells. 2. In differentiated cells, desensitization of 5-hydroxytryptamine (5-HT) responses was much slower than in undifferentiated cells. Desensitization in differentiated cells was also highly variable, with half-times varying by greater than 40-fold. Rapidly desensitized responses in differentiated cells were qualitatively similar to the responses of undifferentiated cells. 3. In outside-out patches from undifferentiated cells, single channel currents could be seen after 5-HT application. These channels had a conductance of 12 pS. The 5-HT-activated channels in differentiated cells were too small to observe at the single-channel level. Noise analysis indicated that the channel conductance was approximately 4 pS. In differentiated cells, both rapidly and slowly desensitized responses were generated by channels with essentially the same conductance. 4. The 5-HT responses of differentiated cells were also distinguished from those of undifferentiated cells on the basis of the voltage dependence of desensitization and the curvature of the current-voltage curve. 5. NG108-15 cells can produce different receptor subtypes, which may be expressed in different tissues or at different stages of development. These variations in receptor behavior suggest that there are at least two distinct mechanisms for regulation of the 5-HT3 receptor.

scholarly journals Mechanism of 4-aminopyridine action on voltage-gated potassium channels in lymphocytes.

1992 ◽  
Vol 99 (2) ◽  
pp. 217-240 ◽  
Author(s):  
D Choquet ◽  
H Korn
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Inactivation Kinetics ◽  
Weak Base ◽  
K Channels ◽  
The Mean ◽  
High Concentrations ◽  
A Site ◽  
Burst Time ◽  
External Ph

The mechanism by which 4-aminopyridine (4-AP) blocks the delayed rectifier type potassium (K+) channels present on lipopolysaccharide-activated murine B lymphocytes was investigated using whole-cell and single channel patch-clamp recordings. 4-AP (1 microM-5 mM) was superfused for 3-4 min before applying depolarizing pulses to activate the channel. During the first pulse after application of 4-AP above 50 microM, the current inactivated faster, as compared with the control, but its peak was only reduced at high concentrations of 4-AP (Kd = 3.1 mM). During subsequent pulses, the peak current was decreased (Kd = 120 microM), but the inactivation rate was slower than in the control, a feature that could be explained by a slow unblocking process. After washing out the drug, the current elicited by the first voltage step was still markedly reduced, as compared with the control one, and displayed very slow activation and inactivation kinetics; this suggests that the K+ channels move from a blocked to an unblocked state slowly during the depolarizing pulse. These results show that 4-AP blocks K+ channels in their open state and that the drug remains trapped in the channel once it is closed. On the basis of the analysis of the current kinetics during unblocking, we suggest that two pathways lead from the blocked to the unblocked states. Computer simulations were used to investigate the mechanism of action of 4-AP. The simulations suggest that 4-AP must bind to both an open and a nonconducting state of the channel. It is postulated that the latter is either the inactivated channel or a site on closed channels only accessible to the drug once the cell has been depolarized. Using inside- and outside-out patch recordings, we found that 4-AP only blocks channels from the intracellular side of the membrane and acts by reducing the mean burst time. 4-AP is a weak base (pK = 9), and thus exists in ionized or nonionized form. Since the Kd of channel block depends on both internal and external pH, we suggest that 4-AP crosses the membrane in its nonionized form and acts from inside the cell in its ionized form.

scholarly journals Slow currents through single sodium channels of the adult rat heart.

1985 ◽  
Vol 86 (1) ◽  
pp. 89-104 ◽  
Author(s):  
J B Patlak ◽  
M Ortiz
Keyword(s):  
Time Constant ◽  
Single Channel ◽  
Slow Component ◽  
Reversal Potential ◽  
Careful Examination ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Adult Rat ◽  
Ventricular Cells ◽  
The Mean

The currents through single Na+ channels from the sarcolemma of ventricular cells dissociated from adult rat hearts were studied using the patch-clamp technique. All patches had several Na+ channels; most had 5-10, while some had up to 50 channels. At 10 degrees C, the conductance of the channel was 9.8 pS. The mean current for sets of many identical pulses inactivated exponentially with a time constant of 1.7 +/- 0.6 ms at -40 mV. Careful examination of the mean currents revealed a small, slow component of inactivation at pulse potentials ranging from -60 to -30 mV. The time constant of the slow component was between 8 and 14 ms. The channels that caused the slow component had the same conductance and reversal potential as the fast Na+ currents and were blocked by tetrodotoxin. The slow currents appear to have been caused by repeated openings of one or more channels. The holding potential influenced the frequency with which such channel reopening occurred. The slow component was prominent during pulses from a holding potential of -100 mV, while it was very small during pulses from -140 mV. Ultraslow currents through the Na+ channel were observed occasionally in patches that had large numbers of channels. They consisted of bursts of 10 or more sequential openings of a single channel and lasted for up to 150 ms. We conclude that the single channel data cannot be explained by standard models, even those that have two inactivated states or two open states of the channel. Our results suggest that Na+ channels can function in several different "modes," each with a different inactivation rate.

scholarly journals Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells

2005 ◽  
Vol 388 (3) ◽  
pp. 941-948 ◽  
Author(s):  
Anna M. BROWN ◽  
Fiona C. RIDDOCH ◽  
Andrew ROBSON ◽  
Christopher P. F. REDFERN ◽  
Timothy R. CHEEK
Keyword(s):  
Retinoic Acid ◽  
Neuronal Differentiation ◽  
Neuroblastoma Cells ◽  
Functional Change ◽  
Plateau Phase ◽  
Functional Changes ◽  
Differentiated Cells ◽  
Fluorescence Measurements ◽  
Undifferentiated Cells ◽  
Ca2 Channels

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.

Variable Regulation of Sensitivity to Retinoic Acid-induced Differentiation in Wild-type and Retinoic Acid-resistant HL-60 Cells

1989 ◽  
Vol 1 (1) ◽  
pp. 45-54 ◽  
Author(s):  
Robert E. Gallagher ◽  
Fernando de Cuevillas ◽  
Chin-Sen Chang ◽  
Edward L. Schwartz
Keyword(s):  
Retinoic Acid ◽  
Wild Type ◽  
Induced Differentiation

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

scholarly journals PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research

2021 ◽  
Vol 11 (9) ◽  
pp. 3729
Author(s):  
Katarzyna Balon ◽  
Benita Wiatrak
Keyword(s):  
Cell Culture ◽  
Dna Damage ◽  
Cell Lines ◽  
Inflammatory Factor ◽  
Research Model ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
Primary Cell Lines ◽  
Neurobiological Research ◽  
The Impact

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS; a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

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