Block of inwardly rectifying K+ currents by extracellular Mg2+ and Ba2+ in bovine pulmonary artery endothelial cells

2000 ◽  
Vol 78 (9) ◽  
pp. 751-756 ◽  
Author(s):  
Yuk Man Leung ◽  
Chiu Yin Kwan ◽  
Edwin E Daniel
Keyword(s):  
Endothelial Cells ◽  
Patch Clamp ◽  
Pulmonary Artery ◽  
Binding Site ◽  
Patch Clamp Technique ◽  
Aortic Endothelial Cells ◽  
Whole Cell Patch Clamp ◽  
Deep Site ◽  
Low Sensitivity ◽  
Inwardly Rectifying

Using whole-cell patch clamp technique, we investigated the blocking effects of extracellular Ba2+ and Mg2+ on the inwardly rectifying K+ (KIR) currents of bovine pulmonary artery endothelial cells (BPAEC). The BPAEC KIR channel has recently been identified as Kir2.1 of the Kir2.0 subfamily. Block of KIR currents by Mg2+ (3-30 mM) was instantaneous, and increased with hyperpolarization slightly (Kd at -160 and 0 mV was 9.5 and 23.2 mM, respectively). The apparent fractional electrical distance (δ) of the Mg2+ binding site is calculated to be 0.07 from the outer mouth of the channel pore. Ba2+ (0.3-10 µM) time-dependently blocked the KIR currents with a much higher potency and stronger voltage-dependence (Kd at -160 and 0 mV was 1.0 and 41.6 µM, respectively). The Ba2+ binding site had a δ value of 0.34. Our data suggest that Mg2+ binds to a very superficial site of the KIR channel, while Ba2+ binds to a much deeper site, sensing much more of the membrane electric field. Thus, the BPAEC Kir2.1 appears to be pharmacologically different from the Kir2.1 reported before in bovine aortic endothelial cells (BAEC), which has 2 sites for Mg2+ block (a deep site in addition to a shallow one), and a superficial and low-sensitivity site for Ba2+ block.Key words: inwardly rectifying K+ channel, patch clamp, Ba2+, Mg2+, endothelial cells.

Inhibition of inwardly rectifying K+ channels by cGMP in pulmonary vascular endothelial cells

2002 ◽  
Vol 283 (2) ◽  
pp. L297-L304 ◽  
Author(s):  
Larissa A. Shimoda ◽  
Laura E. Welsh ◽  
David B. Pearse
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Endothelial Cells ◽  
Cell Types ◽  
Protective Mechanism ◽  
Cell Integrity ◽  
Barrier Dysfunction ◽  
Whole Cell Patch Clamp ◽  
Inward Currents ◽  
Inwardly Rectifying

Endothelial barrier dysfunction is typically triggered by increased intracellular Ca2+concentration. Membrane-permeable analogs of guanosine 3′,5′-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential ( E m), which influences the electrochemical gradient for Ca2+ influx, is regulated by K+ channels, we investigated the effect of 8-bromo-cGMP on E m and inwardly rectifying K+ (KIR) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to −50 mV that were abolished by application of 10 μM Ba2+, consistent with KIR current. Ba2+ also depolarized both cell types. 8-Bromo-cGMP (10−3 M) depolarized BPAEC and BMVEC and inhibited KIR current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of KIR current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for Ca2+ entry.

Molecular mechanisms of action for CFTR potentiators

2019 ◽  
Author(s):  
◽  
Han-I Yeh
Keyword(s):  
Cystic Fibrosis ◽  
Patch Clamp ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Mechanisms Of Action ◽  
Drug Binding ◽  
Patch Clamp Technique ◽  
Chemical Structures ◽  
Clamp Technique ◽  
Sites Of Action

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] As the culprit behind cystic fibrosis (CF) is the dysfunction of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), pharmacological reagents targeting CFTR may hold the key to the ultimate cure of CF. In this thesis, we present the studies in the mechanisms of action for CFTR potentiators, the small molecules that enhance the functions of CFTR. Using the patch-clamp technique, we demonstrated that the permeant anion nitrate modulates CFTR gating through a mechanism similar to the FDA-approved CFTR potentiator VX-770 (ivacaftor). Via separate sites of action, VX-770 and nitrate stabilize the open channel conformation of CFTR in an energetically additive manner. Next, we investigated the action of a novel CFTR potentiator, GLPG1837, and showed that despite their different chemical structures, GLPG1837 and VX-770 share the same mechanisms of action on CFTR gating and compete for a common binding site in the transmembrane domains of CFTR. An allosteric modulation model is further proposed to explain how the affinity and efficacy of both potentiators are determined by the energetic coupling between drug binding and channel gating. Finally, we combined molecular docking and patch-clamp technique to identify the binding site(s) for GLPG1837 and VX-770.

PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes

1998 ◽  
Vol 275 (1) ◽  
pp. C293-C302 ◽  
Author(s):  
Lisa M. Middleton ◽  
Robert D. Harvey
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Perforated Patch Clamp

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.

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.

scholarly journals The Polyamine Binding Site in Inward Rectifier K+ Channels

2006 ◽  
Vol 127 (5) ◽  
pp. 467-480 ◽  
Author(s):  
Harley T. Kurata ◽  
Laurence J. Marton ◽  
Colin G. Nichols
Keyword(s):  
Binding Site ◽  
Exit Rate ◽  
Physical Constraints ◽  
Kir Channel ◽  
Inwardly Rectifying Potassium Channels ◽  
Voltage Dependent ◽  
Deep Site ◽  
Inwardly Rectifying ◽  
Polyamine Binding Site ◽  
Deep Location

Strongly inwardly rectifying potassium channels exhibit potent and steeply voltage-dependent block by intracellular polyamines. To locate the polyamine binding site, we have examined the effects of polyamine blockade on the rate of MTSEA modification of cysteine residues strategically substituted in the pore of a strongly rectifying Kir channel (Kir6.2[N160D]). Spermine only protected cysteines substituted at a deep location in the pore, between the “rectification controller” residue (N160D in Kir6.2, D172 in Kir2.1) and the selectivity filter, against MTSEA modification. In contrast, blockade with a longer synthetic polyamine (CGC-11179) also protected cysteines substituted at sites closer to the cytoplasmic entrance of the channel. Modification of a cysteine at the entrance to the inner cavity (169C) was unaffected by either spermine or CGC-11179, and spermine was clearly “locked” into the inner cavity (i.e., exhibited a dramatically slower exit rate) following modification of this residue. These data provide physical constraints on the spermine binding site, demonstrating that spermine stably binds at a deep site beyond the “rectification controller” residue, near the extracellular entrance to the channel.

scholarly journals Baro-excited neurons in the caudal ventrolateral medulla (CVLM) recorded using the whole-cell patch-clamp technique

2011 ◽  
Vol 35 (5) ◽  
pp. 500-506 ◽  
Author(s):  
Naoki Oshima ◽  
Hiroo Kumagai ◽  
Kamon Iigaya ◽  
Hiroshi Onimaru ◽  
Akira Kawai ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Ventrolateral Medulla ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Caudal Ventrolateral Medulla ◽  
Clamp Technique ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

2002 ◽  
Vol 282 (5) ◽  
pp. H1879-H1888 ◽  
Author(s):  
Regina Macianskiene ◽  
Francesco Moccia ◽  
Karin R. Sipido ◽  
Willem Flameng ◽  
Kanigula Mubagwa
Keyword(s):  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Monovalent Cation ◽  
Cardiac Cells ◽  
Time Dependent ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
High Concentrations ◽  
Low Sensitivity

In cardiac cells that lack macroscopic transient outward K+ currents ( I to), the removal of extracellular Ca2+ can unmask “ I to-like” currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca2+channels underlies these currents. Removal of extracellular Ca2+ and extracellular Mg2+ induced time-independent currents at all potentials and time-dependent currents at potentials greater than −50 mV. Either K+ or Cs+ could carry the time-dependent currents, with reversal potential of +8 mV with internal K+ and +34 mV with Cs+. Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value ( V 1/2) = −24 mV and slope = −9 mV for activation; V 1/2 = −58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC50 = 2 μM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I to-like currents are due to monovalent cation flow through L-type Ca2+ channels, which in pig myocytes show low sensitivity to nifedipine.

scholarly journals Neutrophil Secretion Induced by an Intracellular Ca2+Rise and Followed by Whole-Cell Patch-Clamp Recordings Occurs Without any Selective Mobilization of Different Granule Populations

2006 ◽  
Vol 2006 ◽  
pp. 1-7 ◽  
Author(s):  
Daniel Granfeldt ◽  
Olle Harbecke ◽  
Åse Björstad ◽  
Anna Karlsson ◽  
Claes Dahlgren
Keyword(s):  
Patch Clamp ◽  
Human Neutrophils ◽  
Lag Phase ◽  
Measuring Time ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
High Concentrations ◽  
Kinetics Of

We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance (△Cm). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca2+. A sustained secretion following a short lag phase was induced by high concentrations of Ca2+(100μM and higher). A stable plateau was reached after 5–7 minutes at△Cmvalues corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca2+concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca2+-sensitivity.

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