Extracellular Ba2+ blocks the cardiac transient outward K+ current

2000 ◽  
Vol 278 (1) ◽  
pp. H295-H299 ◽  
Author(s):  
Hong Shi ◽  
Hui-Zhen Wang ◽  
Zhiguo Wang
Keyword(s):  
Patch Clamp ◽  
Inward Rectifier ◽  
Delayed Rectifier ◽  
Inactivation Kinetics ◽  
Atrial Myocytes ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Potent Inhibition ◽  
Voltage Dependent ◽  
K Current

Ba2+ is widely used as a tool in patch-clamp studies because of its ability to block a variety of K+channels and to pass Ca2+ channels. Its potential ability to block the cardiac transient outward K+ current ( I to) has not been clearly documented. We performed whole cell patch-clamp studies in canine ventricular and atrial myocytes. Extracellular application of Ba2+ produced potent inhibition of I to with an IC50 of ∼40 μM. The effects were voltage independent, and the inactivation kinetics were not altered by Ba2+. The potency of Ba2+ was ∼10 times higher than that of 4-aminopyridine (a selective I to blocker with an IC50 of 430 μM) under identical conditions. By comparison, Ba2+blockade of the inward rectifier K+ current was voltage dependent; the IC50 was ∼20 times lower (2.5 μM) than that for I to when determined at −100 mV and was comparable to I to as determined at −60 mV (IC50 = 26 μM). Ba2+ concentrations of ≤1 mM or higher failed to block ultrarapid delayed rectifier K+ current. Our data suggest that Ba2+ can be considered a potent blocker of I to.

scholarly journals Slow inactivation of L-type calcium current distorts the measurement of L- and T-type calcium current in Purkinje myocytes.

1993 ◽  
Vol 102 (5) ◽  
pp. 859-869 ◽  
Author(s):  
N B Datyner ◽  
I S Cohen
Keyword(s):  
Patch Clamp ◽  
Calcium Current ◽  
Slow Inactivation ◽  
Total Calcium ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Method Of Measurement

We have examined slow inactivation of L-type calcium current in canine Purkinje myocytes with the whole cell patch clamp technique. Slow inactivation is voltage dependent. It is negligible at -50 mV but can inactivate more than half of available iCaL at -10 mV. There are two major consequences of this slow inactivation. First, standard protocols for the measurement of T-type current can dramatically overestimate its contribution to total calcium current, and second, the position and steepness of the inactivation versus voltage curve for iCaL will depend on the method of measurement. Given the widespread attempts to identify calcium current components and characterize them biophysically, an important first step should be to determine the extent of slow inactivation of calcium current in each preparation.

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1989 ◽  
Vol 61 (3) ◽  
pp. 467-477 ◽  
Author(s):  
D. E. Meyers ◽  
J. L. Barker
Keyword(s):  
Patch Clamp ◽  
Hippocampal Neurons ◽  
Calcium Currents ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Cells Cultured ◽  
In Cells

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard “run-down” of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the “high-voltage-activated” Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150–450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Voltage-gated transient currents in bovine adrenal fasciculata cells. II. A-type K+ current.

1993 ◽  
Vol 102 (2) ◽  
pp. 239-255 ◽  
Author(s):  
B Mlinar ◽  
J J Enyeart
Keyword(s):  
Hormone Secretion ◽  
Inactivation Kinetics ◽  
Zona Fasciculata ◽  
Time Constants ◽  
Whole Cell ◽  
Type K ◽  
Slow Kinetics ◽  
Boltzmann Function ◽  
Voltage Dependent ◽  
K Current

In whole cell patch clamp recordings on enzymatically dissociated adrenal zona fasciculata (AZF) cells, a rapidly inactivating A-type K+ current was observed in each of more than 150 cells. Activation of IA was steeply voltage dependent and could be described by a Boltzmann function raised to an integer power of 4, with a midpoint of -28.3 mV. Using the "limiting logarithmic potential sensitivity," the single channel gating charge was estimated to be 7.2 e. Voltage-dependent inactivation could also be described by a Boltzmann function with a midpoint of -58.7 mV and a slope factor of 5.92 mV. Gating kinetics of IA included both voltage-dependent and -independent transitions in pathways between closed, open, and inactivated states. IA activated with voltage-dependent sigmoidal kinetics that could be fit with an n4h formalism. The activation time constant, tau a, reached a voltage-independent minimum at potentials positive to 0 mV. IA currents inactivated with two time constants that were voltage independent at potentials ranging from -30 to +45 mV. At +20 mV, tau i(fast) and tau i(slow) were 13.16 +/- 0.64 and 62.26 +/- 5.35 ms (n = 34), respectively. In some cells, IA inactivation kinetics slowed dramatically after many minutes of whole cell recording. Once activated by depolarization, IA channels returned to the closed state along pathways with two voltage-dependent time constants which were 0.208 s, tau rec-f and 10.02 s, tau rec-s at -80 mV. Approximately 90% of IA current recovered with slow kinetics at potentials between -60 and -100 mV. IA was blocked by 4-aminopyridine (IC50 = 629 microM) through a mechanism that was strongly promoted by channel activation. Divalent and trivalent cations including Ni2+ and La3+ also blocked IA with IC50's of 467 and 26.4 microM, respectively. With respect to biophysical properties and pharmacology, IA in AZF cells resembles to some extent transient K+ currents in neurons and muscle, where they function to regulate action potential frequency and duration. The function of this prominent current in steroid hormone secretion by endocrine cells that may not generate action potentials is not yet clear.

Choline chloride activates time-dependent and time-independent K+ currents in dog atrial myocytes

1994 ◽  
Vol 266 (1) ◽  
pp. C42-C51 ◽  
Author(s):  
B. Fermini ◽  
S. Nattel
Keyword(s):  
Choline Chloride ◽  
Outward Current ◽  
Time Dependent ◽  
Equilibrium Potential ◽  
Delayed Rectifier ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
K Current ◽  
K Currents

Using the whole cell configuration of the patch-clamp technique, we studied the effect of isotonic replacement of bath sodium chloride (NaCl) by choline chloride (ChCl) in dog atrial myocytes. Our results show that ChCl triggered 1) activation of a time-independent background current, characterized by a shift of the holding current in the outward direction at potentials positive to the K+ equilibrium potential (EK), and 2) activation of a time- and voltage-dependent outward current, following depolarizing voltage steps positive to EK. Because the choline-induced current obtained by depolarizing steps exhibited properties similar to the delayed rectifier K+ current (IK), we named it IKCh. The amplitude of IKCh was determined by extracellular ChCl concentration, and this current was generally undetectable in the absence of ChCl. IKCh was not activated by acetylcholine (0.001-1.0 mM) or carbachol (10 microM) and could not be recorded in the absence of ChCl or when external NaCl was replaced by sucrose or tetramethylammonium chloride. IKCh was inhibited by atropine (0.01-1.0 microM) but not by the M1 antagonist pirenzepine (up to 10 microM). This current was carried mainly by K+ and was inhibited by CsCl (120 mM, in the pipette) or barium (1 mM, in the bath). We conclude that in dog atrial myocytes, ChCl activates a background conductance comparable to ACh-dependent K+ current, together with a time-dependent K+ current showing properties similar to IK.

Voltage-activated potassium currents of rabbit osteoclasts: effects of extracellular calcium

1994 ◽  
Vol 267 (4) ◽  
pp. C1103-C1111 ◽  
Author(s):  
L. G. Hammerland ◽  
A. S. Parihar ◽  
E. F. Nemeth ◽  
M. C. Sanguinetti
Keyword(s):  
Negative Charge ◽  
Voltage Dependence ◽  
Physiological Function ◽  
Inward Rectifier ◽  
Delayed Rectifier ◽  
Voltage Dependent ◽  
K Current ◽  
Kinetics Of ◽  
K Currents ◽  
Simple Surface

The effects of increased extracellular Ca2+ concentration ([Ca2+]e) were examined on a delayed-rectifier K+ current (IK) and an inward-rectifier K+ current (IK1) in rabbit osteoclasts. Elevation of [Ca2+]e from 1.8 to 18 mM shifted the half point for IK activation by +11.5 mV and the voltage dependence of inactivation by +9.7 mV and slowed the rate of IK activation and deactivation. These effects of elevated [Ca2+]e on IK are consistent with screening of cell surface negative charge. However, elevation of [Ca2+]e increased the voltage-dependent kinetics of IK inactivation at all potentials tested, inconsistent with that predicted by simple surface charge theory. This finding suggests an additional, regulatory role for [Ca2+]e in the gating of IK channels. Some osteoclasts had an IK1, which was decreased when [Ca2+]e was raised from 1.8 to 18 mM. The physiological function of both types of K+ currents remains to be determined, and it is not clear whether these currents are involved with the coupling of cytosolic [Ca2+] to [Ca2+]e.

scholarly journals Kvβ1.3 Reduces the Degree of Stereoselective Bupivacaine Block of Kv1.5 Channels

2007 ◽  
Vol 107 (4) ◽  
pp. 641-651 ◽  
Author(s):  
Cristina Arias ◽  
Miriam Guizy ◽  
Miren David ◽  
Stefanie Marzian ◽  
Teresa González ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Binding Site ◽  
Voltage Clamp ◽  
Xenopus Oocytes ◽  
Time Dependent ◽  
Hek293 Cells ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

Background Kvbeta1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvbeta1.3 competes with them for a binding site at Kv1.5 channels. Methods Currents generated by the activation of Kv1.5 and Kv1.5 + Kvbeta1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. Results Block of Kv1.5, but not that produced on Kv1.5 + Kvbeta1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 microM (n = 15) and 39.8 +/- 8.2 microM (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 microM) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 microM) in Kv1.5 + Kvbeta1.3 channels. Therefore, the degree of stereoselectivity (theta) decreased from 9 to 4 in the presence of Kvbeta1.3. The decrease in potency to block Kv1.5 + Kvbeta1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvbeta1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvbeta1.3 binding) but not for L510A (part of Kvbeta1.3 binding site). Conclusions The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.

scholarly journals Inhibition of cardiac Na+ currents by isoproterenol

1990 ◽  
Vol 258 (4) ◽  
pp. H977-H982 ◽  
Author(s):  
B. Schubert ◽  
A. M. Vandongen ◽  
G. E. Kirsch ◽  
A. M. Brown
Keyword(s):  
Patch Clamp ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Na Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Voltage Dependent

The mechanism by which the beta-adrenergic agonist isoproterenol (ISO) modulates voltage-dependent cardiac Na+ currents (INa) was studied in single ventricular myocytes of neonatal rat using the gigaseal patch-clamp technique. ISO inhibited INa reversibly, making the effect readily distinguishable from the monotonic decrease of INa caused by the shift in gating that customarily occurs during whole cell patch-clamp experiments (E. Fenwick, A. Marty, and E. Neher, J. Physiol. Lond. 331: 599-635, 1982; and J. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. Lond. 356: 565-585, 1984). The inhibition was biphasic, having fast and slow components, and was voltage-dependent, being more pronounced at depolarized potentials. In whole cell experiments the membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) congener 8-bromo-cAMP reduced INa. In cell-free inside-out patches with ISO present in the pipette, guanosine 5'-triphosphate (GTP) applied to the inner side of the membrane patch inhibited single Na+ channel activity. This inhibition could be partly reversed by hyperpolarizing prepulses. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) greatly reduced the probability of single Na+ channel currents in a Mg2(+)-dependent manner. We propose that ISO inhibits cardiac Na+ channels via the guanine nucleotide binding, signal-transducing G protein that acts through both direct (membrane delimited) and indirect (cytoplasmic) pathways.

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