scholarly journals Usefulness and Limitation of DiBAC4(3), a Voltage-Sensitive Fluorescent Dye, for the Measurement of Membrane Potentials Regulated by Recombinant Large Conductance Ca2+-Activated K+ Channels in HEK293 Cells

2001 ◽  
Vol 86 (3) ◽  
pp. 342-350 ◽  
Author(s):  
Aki Yamada ◽  
Norikazu Gaja ◽  
Susumu Ohya ◽  
Katsuhiko Muraki ◽  
Hiroshi Narita ◽  
...  
Keyword(s):  
Membrane Potentials ◽  
Fluorescent Dye ◽  
Hek293 Cells ◽  
K Channels

scholarly journals Saxitoxin Is a Gating Modifier of hERG K+ Channels

2003 ◽  
Vol 121 (6) ◽  
pp. 583-598 ◽  
Author(s):  
Jixin Wang ◽  
Joseph J. Salata ◽  
Paul B. Bennett
Keyword(s):  
Channel Gating ◽  
Cellular Membrane ◽  
Membrane Potentials ◽  
Hek293 Cells ◽  
K Channel ◽  
K Channels ◽  
Channel Opening ◽  
Kinetics Of ◽  
K Currents ◽  
Voltage Activation

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent “agonist-like” effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels

1992 ◽  
Vol 68 (4) ◽  
pp. 985-1000 ◽  
Author(s):  
H. Sontheimer ◽  
J. A. Black ◽  
B. R. Ransom ◽  
S. G. Waxman
Keyword(s):  
Spinal Cord ◽  
Membrane Potentials ◽  
Resting Potential ◽  
K Channel ◽  
Na Channels ◽  
Patch Clamp Recording ◽  
K Channels ◽  
Na Currents ◽  
Channel Expression

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.

scholarly journals Inactivation Gating of Kv4 Potassium Channels

1999 ◽  
Vol 113 (5) ◽  
pp. 641-660 ◽  
Author(s):  
Henry H. Jerng ◽  
Mohammad Shahidullah ◽  
Manuel Covarrubias
Keyword(s):  
Time Course ◽  
Membrane Potentials ◽  
Structural Basis ◽  
K Channel ◽  
Inactivation Curve ◽  
Double Mutation ◽  
K Channels ◽  
Closed State ◽  
Recovery From Inactivation ◽  
Inactivation Gating

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance–voltage curve (∼5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specifc residue in the S4–S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404,406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.

Monitoring membrane potentials in Erhlich ascites tumor cells by means of a fluorescent dye

1976 ◽  
Vol 436 (2) ◽  
pp. 475-488 ◽  
Author(s):  
Philip C. Laris ◽  
Harrihar A. Pershadsingh ◽  
Rose M. Johnstone
Keyword(s):  
Tumor Cells ◽  
Membrane Potentials ◽  
Fluorescent Dye ◽  
Ascites Tumor

scholarly journals Kv11 ( ether‐à‐go‐go ‐related gene) voltage‐dependent K + channels promote resonance and oscillation of subthreshold membrane potentials

2020 ◽  
Author(s):  
Toshinori Matsuoka ◽  
Miwako Yamasaki ◽  
Manabu Abe ◽  
Yukiko Matsuda ◽  
Hiroyuki Morino ◽  
...  
Keyword(s):  
Membrane Potentials ◽  
Related Gene ◽  
K Channels ◽  
Voltage Dependent

scholarly journals Validation of a [3H]Astemizole Binding Assay in HEK293 Cells Expressing HERG K+ Channels

2004 ◽  
Vol 95 (3) ◽  
pp. 311-319 ◽  
Author(s):  
Peter J.S. Chiu ◽  
Karen F. Marcoe ◽  
Sidney E. Bounds ◽  
Chun-Hsiung Lin ◽  
Jin-Jye Feng ◽  
...  
Keyword(s):  
Binding Assay ◽  
Hek293 Cells ◽  
K Channels

scholarly journals ACTH Inhibits bTREK-1 K+ Channels through Multiple cAMP-dependent Signaling Pathways

2008 ◽  
Vol 132 (2) ◽  
pp. 279-294 ◽  
Author(s):  
Haiyan Liu ◽  
Judith A. Enyeart ◽  
John J. Enyeart
Keyword(s):  
Hek293 Cells ◽  
Resting Membrane Potential ◽  
Zona Fasciculata ◽  
Nucleotide Exchange ◽  
Pipette Solution ◽  
K Channels ◽  
Whole Cell Patch Clamp ◽  
Dependent Protein ◽  
Secretion Inhibition ◽  
And Function

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels that set the resting membrane potential and function pivotally in the physiology of cortisol secretion. Inhibition of these K+ channels by adrenocorticotropic hormone (ACTH) or cAMP is coupled to depolarization and Ca2+ entry. The mechanism of ACTH and cAMP-mediated inhibition of bTREK-1 was explored in whole cell patch clamp recordings from AZF cells. Inhibition of bTREK-1 by ACTH and forskolin was not affected by the addition of both H-89 and PKI(6–22) amide to the pipette solution at concentrations that completely blocked activation of cAMP-dependent protein kinase (PKA) in these cells. The ACTH derivative, O-nitrophenyl, sulfenyl-adrenocorticotropin (NPS-ACTH), at concentrations that produced little or no activation of PKA, inhibited bTREK-1 by a Ca2+-independent mechanism. Northern blot analysis showed that bovine AZF cells robustly express mRNA for Epac2, a guanine nucleotide exchange protein activated by cAMP. The selective Epac activator, 8-pCPT-2′-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC50 = 0.63 μM) at concentrations that did not activate PKA. Inhibition by this agent was unaffected by PKA inhibitors, including RpcAMPS, but was eliminated in the absence of hydrolyzable ATP. Culturing AZF cells in the presence of ACTH markedly reduced the expression of Epac2 mRNA. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 current in AZF cells that had been treated with ACTH for 3–4 d while inhibition by 8-br-cAMP was not affected. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 expressed in HEK293 cells, which express little or no Epac2. These findings demonstrate that, in addition to the well-described PKA-dependent TREK-1 inhibition, ACTH, NPS-ACTH, forskolin, and 8-pCPT-2′-O-Me-cAMP also inhibit these K+ channels by a PKA-independent signaling pathway. The convergent inhibition of bTREK-1 through parallel PKA- and Epac-dependent mechanisms may provide for failsafe membrane depolarization by ACTH.

Normal function of HERG K+ channels expressed in HEK293 cells requires basal protein kinase B activity

FEBS Letters ◽  
2002 ◽  
Vol 534 (1-3) ◽  
pp. 125-132 ◽  
Author(s):  
Yiqiang Zhang ◽  
Huizhen Wang ◽  
Jingxiong Wang ◽  
Hong Han ◽  
Stanley Nattel ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Normal Function ◽  
Hek293 Cells ◽  
K Channels

scholarly journals Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel.

1993 ◽  
Vol 102 (4) ◽  
pp. 713-728 ◽  
Author(s):  
L D Chabala ◽  
N Bakry ◽  
M Covarrubias
Keyword(s):  
Molecular Weight ◽  
Xenopus Oocytes ◽  
Membrane Potentials ◽  
K Channel ◽  
Low Molecular Weight ◽  
K Channels ◽  
Fourfold Increase ◽  
Mrna Species ◽  
Type K

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.

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