P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells

1997 ◽  
Vol 273 (6) ◽  
pp. C1793-C1800 ◽  
Author(s):  
Robson Coutinho-Silva ◽  
Pedro Muanis Persechini
Keyword(s):  
Molecular Weight ◽  
Bone Marrow ◽  
Patch Clamp ◽  
Plasma Membranes ◽  
Extracellular Atp ◽  
Low Molecular Weight ◽  
Whole Cell ◽  
Voltage Dependent ◽  
J774 Cells ◽  
Bone Marrow Derived Cells

Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 ± 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-d-glucamine, and glutamate; their opening is favored at temperatures higher than 30°C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3′- O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.

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.

Trypsin-uncoupled synthesis and secretion of yeast invertase: implications for the mechanism of secretion

1979 ◽  
Vol 25 (4) ◽  
pp. 528-534 ◽  
Author(s):  
Bruce E. Holbein ◽  
Denis K. Kidby
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
External Surface ◽  
Internal Surface ◽  
Low Molecular Weight ◽  
Synthetase Activity ◽  
Trypsin Treatment ◽  
Yeast Invertase

The subcellular distribution of invertase was examined after synthesis and secretion by sphaeroplasts had been uncoupled by the addition of 30 μg mL−1 trypsin. Sphaeroplasts secreted only the high molecular weight invertase during uncoupling by trypsin. The level of low molecular weight, 'small' invertase in the soluble internal pool was found to be elevated by over fivefold, and the membrane-associated pool was found to contain low molecular weight invertase in addition to intermediate molecular weight invertase, after 1.5 h of trypsin treatment. Purified plasma membranes from trypsin-treated sphaeroplasts had no detectable mannan synthetase activity. On the basis of these and previous findings, a working hypothesis wherein invertase is synthesized on the internal surface of the plasma membrane and glycosylated during its transit to the external surface is presented.

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)

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