Endogenous Kv channels in human embryonic kidney (HEK-293) cells.
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HEK 293 cells
Tetraethylammonium chloride
4-Aminopyridine
Niflumic acid
Potassium channel blocker
HEK 293 cells
Tetraethylammonium chloride
4-Aminopyridine
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Potassium channel blocker
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Objective To investigate the endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells. Methods The standard whole-cell recording method was used to record different outward currents in HEK293 cells. And the detailed electrophysiology of these channels were studied. Results No obvious chloride currents were found in native HEK293 cells. No obvious background potassium currents or delayed rectifier potassium currents were recorded either. 4-aminopyridine (4-AP) sensitive potas- sium currents were found in 59.7% of HEK293 cells. 2 mmol/L 4-AP can completely inhibit this current. The amplitude of this current was (175±112)pA at 40mV. The inactivation curve shows the V1/2 was (-3.5±0.9) mV,with a slope of (6.3±0.8)mV. The inactivation kinetics of this channel was bell-shaped. The recovery time constant of this channel from inactivation was (333.3±33.9)ms. All these data suggest this current is similar to transient outward potassium current (Ito) and this channel is the main endogenous functional channel in HEK293 cells. Conclusion Transient outward potassium current exists in native HEK293 cells, which is a widely used expressing system of different protein including channel protein. So we should be cautious in explaining the results of the expressed channels in HEK293 cells. A control should be necessary.
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1. The relationship between ionic current inactivation and immobilization of ‘off'‐gating charge in human Kv1.5 channels expressed in human embryonic kidney (HEK293) cells was studied using 4‐aminopyridine (4‐AP) and tetraethylammonium chloride (TEA‐Cl). 2. The charge transferred during short (< 10 ms) depolarizations (Q(on)) was conserved on repolarization (Q(off)) although peak off‐gating current (off‐Ig) was reduced and the time course prolonged (tau decay increased from 0.4 to > 1.2 ms). For +80 mV pulses longer than 50 ms, Q(off) at 20 ms was less than Q(on) (Q(off)/Q(on) ratio was 0.26 +/‐ 0.06 at 450 ms). We attribute this to a relative ‘immobilization’ of gating charge during long depolarizations. 3. 4‐AP (0.1‐1 mM) prevented slowing of off‐Ig, allowing saturation of peak off‐Ig. 4‐AP also completely prevented immobilization of off‐Ig after long depolarizations. In 1 mM 4‐AP, off‐Ig waveforms decayed rapidly and the charge ratio Q(off)/Q(on) remained at 1.0. 4. In addition to its effects on Ig, 1 mM 4‐AP prevented the slow inactivation of ionic current seen during strong depolarizations. An initial block was caused by 4‐AP or 1 mM intracellular TEA internally applied. However, only 4‐AP prevented the slower, later development of C‐type inactivation. 5. We suggest that slow current inactivation is accompanied by a gating charge immobilization in Kv1.5. 4‐AP potently inhibits the changes in Q(off)/Q(on0, off‐Ig, and ionic currents that underlie slow inactivation. Some actions of 4‐AP appear independent of its properties as a blocker of open K+ channels, and are not mimicked by internal TEA.
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The effects of aminopyridine analogs on Ca2+-activated K+ channels in GH3 clonal anterior pituitary cells were studied using whole-cell voltage-clamp and single-channel recording techniques. Step depolarization from a holding potential of -50 mV activated a noninactivating, tetraethylammonium- and Cd2+-sensitive outward current. Tail current analysis indicated that this sustained outward current is carried predominantly by K+ ions. Extracellular perfusion with 4-aminopyridine and 3,4-diaminopyridine (0.05-5 mM) caused a dose-dependent enhancement of the outward current by up to 100 and 170%, respectively. This effect typically occurred with prolonged depolarizations of greater than 1-2 sec. Patch-clamp recordings in the cell-attached configuration demonstrated that 4-aminopyridine (2 mM) promotes the activity of a large-conductance (150-175 pS; 50-135 mM external K+), tetraethylammonium-sensitive, Ca2+-activated K+ channel; the drug had no effect on these channels in excised patches. These results indicate that aminopyridines enhance the opening of Ca2+-activated K+ channels in GH3 cells. Several lines of evidence suggest that this effect may occur indirectly, possibly as a result of an increase in the effective intracellular free Ca2+ level.
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Objective: To investigate the effect of HERG(Human ether-a-go-go related gene) potassium channels on the regulatory volume decrease and to reveal the possible mechanism. Methods: The stable HERG-transfected HEK293 cell line and HERG non-transfected HEK293 cell line were used for all the experiments, HERG potassium current was recorded in whole-cell patch-clamp techniques. Results: 1 When the HERG-HEK293 cells are exposed to the hypotonic solution and the test potential is 0mV, the Istep increased 60%(n=12, P0.05); the Itail increased 72.1%(n=11, P0.01); the increase currents can be inhibited by the specific blocker Cisapride(100nmol/L), Istep can be inhibited by 97.2%(n=6, P0.01) and the Itail can be inhibited by 174.1%(n=6, P0.01). 2Exposure to the hypotonic solution and in the presence of the volume regulated chloride channel blockers Niflumic Acid(10 nmol/L), the Istep can be inhibited by 46.2%(n=12, P0.01) and the Itail can be inhibited by 48.5%(n=11, P0.01); In the presence of DIDS(100μmol/L), the Istep can be inhibited by 45.9%(n=12, P0.01) and Itail can be inhibited by 51.1%(n=11, P0.01). Conclusion: HERG potassium channels are partly involved in the process of the regulatory volume decrease, and the involvement followed the activation of the volume regulated chloride channel.
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Background:Dalfampridine (4-aminopyridine; 4-AP) is a potassium channel blocker that has been available in the United States as a treatment to improve walking in patients with multiple sclerosis. 4-AP is well-characterized in vitro with regard to inhibition of neuronal potassium channels, but the potential contribution of its metabolites to clinical activity has not been determined. This study evaluated the concentration–response of 4-AP and its two primary metabolites, 3-hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate, for inhibition of the potassium channels Kv 1.1, Kv 1.2, and Kv 1.4, which are considered candidates for mediating effects of 4-AP on action potential conduction because of their presence in axonal membranes.Methods:Stable transfection of cDNA for Kv 1.1, Kv 1.2, and Kv 1.4 was performed into HEK293 cells, and colonies of cells containing each channel were selected and maintained under appropriate cell culture conditions. Electrophysiological measurements were performed using a patch-clamp technique in at least three cells for each concentration (50, 500, 5000, and 50,000 μM) of 4-AP and the two metabolites, with each cell acting as its own control. Concentration–response curves were constructed for 4-AP and each metabolite. Data were analyzed using nonlinear least-squares fit, and concentrations inhibiting the channels by 50% (IC50) were estimated.Results:4-AP induced similar concentration-dependent inhibition profiles of all three potassium channels, resulting in a narrow range of IC50 values across channels (242 µM to 399 µM). Across the three channels, the IC50 values of 3-hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate were 1–2 orders of magnitude higher (less potent) than those of 4-AP.Conclusions:3-Hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate demonstrated low in vitro potency for Kv 1.1, Kv 1.2, and Kv 1.4 inhibition, suggesting that these metabolites are unlikely to contribute to the positive pharmacodynamic effects of 4-AP. A limitation of this study is that while the metabolites were substantially less active at these representative potassium channels in vitro, the untested possibility exists that they may be active at one or more of the many other channel types that occur in vivo.
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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.
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AIM: To establish a cell line stably expressing human cardiac Kv1.5 potassium channel. METHODS: Human cardiac hKv1.5 gene was transfected into HEK 293 cells. G418 at 1 mg/mL was used to select the transfected cells. After 2 weeks for selection, the anti-G418 cell clones were further identified with patch-clamp method, and whole cell currents were recorded. RESULTS: hKv1.5 gene was successfully transfected into HEK 293 cells. The expressed hKv1.5 currents were ultra-rapid outward currents with obvious voltage-dependence. Its average amplitude was (3.20±0.60) nA and its activation voltage was about -30 mV. CONCLUSION: The cell line stably expressing human atrial Kv1.5 channels has been successfully established, which has the similar characteristics of human atrial I Kur. The established cell line can be used as a cell model for studying in vitro human atrial I Kur.
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We have investigated the influence of voltage-dependent, potassium conductances on the migration of embryonic neurons, using a culture preparation taken from the acoustico-vestibular anlage long before the onset of electrical excitability and synaptic function. Whole-cell patch clamp recordings from migrating neuroblasts at Hamburger-Hamilton stage 28 (E 5.5) revealed the exclusive expression of voltage-dependent, high-threshold, outward currents, activating at potentials positive to -20 mV. These currents were completely suppressed by the potassium channel blockers, 1.0 mM tetraethylammonium chloride (TEA) or 1.0 mM 4-aminopyridine (4-AP). In control media, the active migration of individual neuroblasts was recorded at 27 ± 6 μm per hr. Within minutes after adding either drug to the culture, normal migration completely stopped for several hours. Calcium channel blockers, ω-conotoxin (3 μM) or cadmium chloride (100 μM), slowed, but did not halt, migration, while nickel chloride (100 μM) or N-methyl-D-glucamine (1 mM) had no effect. However, within 8 hr after TEA exposure, migratory activity usually returned. This recovery was associated with the appearance of a previously undetected, low-threshold and 4-AP- sensitive potassium conductance. We suggest that high-threshold, TEA/4-AP-sensitive potassium channels may normally support the migration of these neurons, while their chronic blockade can be compensated by the appearance of novel potassium channels. Potassium currents may act in concert with N-type calcium channels to regulate neuronal migration. J. Neurosci. Res. 58:805–814, 1999. © 1999 Wiley-Liss, Inc.
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