Kinetic and pharmacological properties of low voltage-activated Ca2+ current in rat clonal (GH3) pituitary cells

1992 ◽  
Vol 68 (1) ◽  
pp. 213-232 ◽  
Author(s):  
J. Herrington ◽  
C. J. Lingle
Keyword(s):  
Time Course ◽  
Low Voltage ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Anesthetic Agents ◽  
Channel Opening ◽  
Cell Recording ◽  
Voltage Dependent ◽  
Dependent Processes ◽  
Site Binding

1. Low voltage-activated (LVA) Ca2+ current in clonal (GH3) pituitary cells was studied with the use of the whole-cell recording technique. The use of internal fluoride to facilitate the rundown of high voltage-activated (HVA) Ca2+ current allowed the study of LVA current in virtual isolation. 2. In 10 mM [Ca2+]o, detectable LVA current begins to appear at about -50 mV, with half-maximal activation occurring at -33 mV. The time course of activation was best described by a Hodgkin-Huxley expression with n = 3, suggesting that at least three closed states must be traversed before channel opening. 3. Deactivation was found to vary exponentially with membrane potential between -60 and -160 mV, indicating that channel closing is rate-limited by a single, voltage-dependent transition. 4. Onset and removal of inactivation between -40 and -130 mV were best described by the sum of two exponentials. Between -80 and -130 mV, both components of removal of inactivation showed little voltage dependence, with time constants of approximately 200-300 ms and 1-2 s. At membrane potentials above -40 mV, a single component of inactivation onset was detected. This component was voltage independent between -20 and +20 mV (tau = 22 ms). Thus inactivation of LVA current is best described by multiple, voltage-in-dependent processes. 5. Significant inactivation of LVA current occurred at -65 mV without detectable macroscopic current. This suggests that inactivation is not strictly coupled to channel opening. 6. Peak LVA current increased with increasing [Ca2+]o, with saturation approximately 50 mM. The Ca(2+)-dependence of peak LVA current was reasonably well described by a single-site binding isotherm with half-maximal LVA current at approximately 7 mM. 7. LVA current in GH3 cells was largely resistant to blockade by Ni2+. The relative potency of inorganic cations in blocking GH3 LVA current was (concentrations which produced 50% block): La3+ (2.4 microM) greater than Cd2+ (188 microM) greater than Ni2+ (777 microM). 8. Several organic agents, including putative LVA blockers, HVA current blockers and various anesthetic agents, were tested for their ability to block LVA current. The concentrations that produced 50% block are as follows: nifedipine (approximately 50 microM), D600 (51 microM), diltiazem (131 microM), octanol (244 microM), pentobarbital (985 microM), methoxyflurane (1.41 mM), and amiloride (1.55 mM). Phenytoin and ethosuximide produced 36 and 10% block at 100 microM and 2.5 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent.

1989 ◽  
Vol 94 (2) ◽  
pp. 213-232 ◽  
Author(s):  
G Cota ◽  
C M Armstrong
Keyword(s):  
Time Course ◽  
Inactivation Rate ◽  
Gh3 Cells ◽  
Na Channel ◽  
Pituitary Cells ◽  
Channel Inactivation ◽  
Internal Medium ◽  
Whole Cell Patch Clamp ◽  
Before And After ◽  
Voltage Dependent

We have determined the time course of Na channel inactivation in clonal pituitary (GH3) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole-cell patch clamp, with papain included in the internal medium. Inactivation was slowly removed over the course of 10 min, making it possible to obtain control records before the enzyme acted. Papain caused a large (4-100x) increase in current magnitude for small depolarizations (near -40 mV), and a much smaller increase for large ones (approximately 1.5x at +40 mV). For technical reasons it was sometimes convenient to study outward INa recorded with no Na+ outside. The instantaneous I-V (IIV) curve in this condition was nonlinear before papain, and more nearly linear afterwards. The gNa-V curve after papain, obtained by dividing the INa-V curve by the IIV curve, was left-shifted by at least 20 mV and steepened. A spontaneous 5-10 mV left shift occurred in the absence of papain. The rate of the inactivation step was found to vary only slightly from -100 mV to +60 mV, based on the following evidence. (a) Before papain, inactivation rate saturated with voltage and was constant from +20 to +60 mV. (b) We activated the channels with a brief pulse, and studied the time course of the current on changing the voltage to a second, usually more negative level (Na+ present internally and externally). The time course of inactivation at each voltage was obtained by comparing control traces with those after inactivation was removed. When the 5-10-mV spontaneous shift was taken into account, inactivation rate changed by less than 10% from -100 to +60 mV. The data are considered in terms of existing models of the Na channel.

scholarly journals Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells.

1988 ◽  
Vol 91 (6) ◽  
pp. 817-833 ◽  
Author(s):  
P A Pappone ◽  
M T Lucero
Keyword(s):  
Potassium Channels ◽  
Potassium Channel ◽  
Potassium Current ◽  
Potassium Currents ◽  
Scorpion Venom ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Crude Venom ◽  
Voltage Gated ◽  
Voltage Dependent

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.

Low-voltage activated calcium currents increase in basal forebrain neurons from aged rats

1995 ◽  
Vol 74 (2) ◽  
pp. 876-887 ◽  
Author(s):  
D. Murchison ◽  
W. H. Griffith
Keyword(s):  
Time Course ◽  
Low Voltage ◽  
Age Groups ◽  
Medial Septum ◽  
Calcium Currents ◽  
Fischer 344 ◽  
Diagonal Band ◽  
Whole Cell Patch Clamp ◽  
Age Related ◽  
Voltage Dependent

1. Whole cell patch-clamp recordings were made of low-voltage-activated (LVA) calcium (Ca2+) currents using 2 mM barium (Ba2+) as charge carrier. Acutely dissociated neurons from medial septum (MS) and the nucleus of the diagonal band (nDB) were examined in young adult (1–3 mo) and aged (24–26 mo) Fischer 344 rats. 2. Most neurons in both age groups displayed LVA currents: 84% of young cells (110/131) and 87% in aged cells (62/71). Using cell capacitance as an indication of cell size, aged cells were significantly smaller (P < 0.05; 15.4 +/- 0.6 pF; mean +/- SE) than young cells (18.0 +/- 0.5 pF), although a single distribution of cell sizes was present in both populations. 3. The LVA currents were enhanced in cells from aged animals. When LVA currents were studied without activation of high voltage activated currents, the current density (pA/pF) was significantly (P < 0.05) increased at negative potentials in aged neurons (young: 4.92 +/- 0.35 pA/pF; Aged: 5.92 +/- 0.45 pA/pF, at a prepulse potential of -110 mV). No change in voltage-dependent activation or inactivation was seen. The time course of recovery from inactivation also was unchanged. 4. Kinetic parameters of LVA currents were compared in both age groups. No age-related difference in time-dependent activation or inactivation was observed. A single distribution of decay time constants of LVA currents was present in both age groups. 5. These results show that MS/nDB cells maintain robust LVA currents and have increased current densities in very old rats. An increased LVA current in the aged neurons suggests that their ability to fire rhythmically or in bursts is retained or enhanced and that the resulting increase in intracellular Ca2+ may contribute to an altered Ca2+ homeostasis.

scholarly journals Desensitization to ANG II in guinea pig ileum depends on membrane repolarization: role of maxi-K+ channel

1999 ◽  
Vol 277 (4) ◽  
pp. C739-C745 ◽  
Author(s):  
Bagnólia A. Silva ◽  
Viviane L. A. Nouailhetas ◽  
Jeannine Aboulafia
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
K Channel ◽  
Ang Ii ◽  
Guinea Pig Ileum ◽  
Tonic Component ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Sustained Activation

Desensitization of ANG II tonic contractile response of the guinea pig ileum is related to membrane repolarization determined by Ca2+-activated K+(maxi-K+) channel opening. ANG II-stimulated depolarized myocytes presented sustained activation of maxi-K+ channels, characterized by reduction from 415 to 12 ms of the closed time constant. ANG II desensitization was prevented by 100 nM iberiotoxin, being reversible within 30 min. Depolarization by KCl, higher than 4 mM, impaired desensitization, suggesting that the membrane potential must attain a threshold to counteract the repolarization induced by maxi-K+ channel opening. Once this value is attained, there is no time dependency because the desensitization process was shut off by addition of KCl along the time course of the tonic response. In contrast, the sustained ACh tonic component was not altered by these maneuvers. We conclude that desensitization of the ANG II tonic component is foremost due to the opening of maxi-K+ channels, leading to membrane repolarization, thus closing the voltage-dependent Ca2+ channels responsible for the Ca2+ influx that sustains the tonic component in this muscle.

Activation and Deactivation of Voltage-Dependent K+ Channels During Synaptically Driven Action Potentials in the MNTB

2006 ◽  
Vol 96 (3) ◽  
pp. 1547-1555 ◽  
Author(s):  
Achim Klug ◽  
Laurence O. Trussell
Keyword(s):  
Action Potentials ◽  
Time Course ◽  
Low Voltage ◽  
Multiple Roles ◽  
Synaptic Activity ◽  
Leak Current ◽  
Medial Nucleus ◽  
Voltage Dependent ◽  
Spike Waveforms ◽  
Trapezoid Body

K+ channels shape individual action potentials and determine their pattern of firing. In auditory relays, both high- and low-voltage–activated K+ channels (HVA and LVA) are critical for preservation of auditory timing cues. We examined how these channels participate in firing in the medial nucleus of the trapezoid body. Principal cells at physiological temperature were voltage clamped using spike waveforms previously recorded in response to calyceal firing. Current components were isolated by digital subtraction of traces recorded in the channel antagonists dendrotoxin-I or tetraethylammonium. During orthodromic spikes delivered at 300 and 600 Hz, both currents activated with a slight delay, peaking just after the crest of the spike. The decay of HVA was sufficiently fast to match the time course of the spike. By contrast, with 300-Hz stimuli, LVA continued to decay after the spikes reached a stable interspike potential. Although LVA currents partially inactivate during prolonged voltage steps, their peak amplitudes remained stable or increased during trains of spikelike stimuli. At 600 Hz, LVA did not fully deactivate between the spikes and therefore generated a leak current. To determine the effect of blocking LVA channels on spiking, prerecorded postsynaptic conductances were injected, with and without dendrotoxin-I. After block of LVA channels, strong synaptic conductances produced broader spikes, greater spike jitter, and prolonged depolarized states. HVA blockade with tetraethylammonium also broadened spikes but led to less error in timing. These results reveal multiple roles for LVA channels in spike repolarization and timing during synaptic activity.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

scholarly journals Minireview: Aldosterone Biosynthesis: Electrically Gated for Our Protection

Endocrinology ◽  
2012 ◽  
Vol 153 (8) ◽  
pp. 3579-3586 ◽  
Author(s):  
Nick A. Guagliardo ◽  
Junlan Yao ◽  
Changlong Hu ◽  
Paula Q. Barrett
Keyword(s):  
Time Course ◽  
Low Voltage ◽  
Salt Water ◽  
Zona Glomerulosa ◽  
Membrane Voltage ◽  
Disease States ◽  
Voltage Dependent ◽  
Aldosterone Production ◽  
Ca2 Channels

Aldosterone produced by adrenal zona glomerulosa (ZG) cells plays an important role in maintaining salt/water balance and, hence, blood pressure homeostasis. However, when dysregulated, aldosterone advances renal and cardiovascular disease states. Multiple steps in the steroidogenic pathway require Ca2+, and the sustained production of aldosterone depends on maintained Ca2+ entry into the ZG cell. Nevertheless, the recorded membrane potential of isolated ZG cells is extremely hyperpolarized, allowing the opening of only a small fraction of low-voltage-activated Ca2+ channels of the Cav3.x family, the major Ca2+ conductance on the ZG cell membrane. As a consequence, to activate sufficient Ca2+ channels to sustain the production of aldosterone, aldosterone secretagogs would be required to affect large decreases in membrane voltage, a requirement that is inconsistent with the exquisite sensitivity of aldosterone production in vivo to small changes (0.1 mm) in extracellular K+. In this review, we evaluate the contribution of membrane voltage and voltage-dependent Ca2+ channels to the control of aldosterone production and consider data highlighting the electrical excitability of the ZG cell. This intrinsic capacity of ZG cells to behave as electrical oscillators provides a platform from which to generate a recurring Ca2+ signal that is compatible with the lengthy time course of steroidogenesis and provides an alternative model for the physiological regulation of aldosterone production that permits both amplitude and temporal modulation of the Ca2+ signal.

scholarly journals Inactivation viewed through single sodium channels.

1984 ◽  
Vol 84 (4) ◽  
pp. 535-564 ◽  
Author(s):  
C A Vandenberg ◽  
R Horn
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Gh3 Cells ◽  
Activation Process ◽  
Whole Cell ◽  
Open Time ◽  
Voltage Dependent

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.

scholarly journals Characterization of the Inhibitory Effect of Gastrodigenin and Gastrodin on M-type K+ Currents in Pituitary Cells and Hippocampal Neurons

2019 ◽  
Vol 21 (1) ◽  
pp. 117 ◽  
Author(s):  
Chih-Sheng Yang ◽  
Ming-Chi Lai ◽  
Ping-Yen Liu ◽  
Yi-Ching Lo ◽  
Chin-Wei Huang ◽  
...  
Keyword(s):  
Current Density ◽  
Time Course ◽  
Gh3 Cells ◽  
Delayed Rectifier ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Activation Time ◽  
Patch Clamp Technique ◽  
Type K ◽  
K Current

Gastrodigenin (HBA) and gastrodin (GAS) are phenolic ingredients found in Gastrodia elata Blume (GEB), a traditional Chinese herbal medicine. These compounds have been previously used to treat cognitive dysfunction, convulsion, and dizziness. However, at present, there is no available information regarding their potential ionic effects in electrically excitable cells. In the current study, the possible effects of HBA and GAS on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were investigated using the patch-clamp technique. The addition of HBA or GAS resulted in the differential inhibition of the M-type K+ current (IK(M)) density in a concentration-dependent manner in GH3 cells. HBA resulted in a slowing of the activation time course of IK(M), while GAS elevated it. HBA also mildly suppressed the density of erg-mediated or the delayed-rectifier K+ current in GH3 cells. Neither GAS nor HBA (10 µM) modified the voltage-gated Na+ current density, although they suppressed the L-type Ca2+ current density at the same concentration. In hippocampal mHippoE-14 neurons, HBA was effective at inhibiting IK(M) density as well as slowing the activation time course. Taken together, the present study provided the first evidence that HBA or GAS could act on cellular mechanisms, and could therefore potentially have a functional influence in various neurologic disorders.

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