Are second messengers crucial for opening the pore associated with P2X7receptor?

2005 ◽  
Vol 288 (2) ◽  
pp. C260-C271 ◽  
Author(s):  
R. X. Faria ◽  
F. P. DeFarias ◽  
Luiz Anastácio Alves
Keyword(s):  
Cell Membrane ◽  
Pore Formation ◽  
Single Channel ◽  
Second Messengers ◽  
Second Messenger ◽  
Fluorescent Imaging ◽  
Cationic Channels ◽  
Intracellular Ca ◽  
Single Channel Currents ◽  
Channel Currents

Stimulation of the P2X7receptor by ATP induces cell membrane depolarization, increase in intracellular Ca2+concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X7, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X7pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X7pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X7receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca2+as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca2+and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X7pore depends on second messengers such as Ca2+and MAP kinases.

scholarly journals A comparison of calcium-activated potassium channel currents in cell-attached and excised patches.

1987 ◽  
Vol 89 (6) ◽  
pp. 985-997 ◽  
Author(s):  
B S Pallotta ◽  
J R Hepler ◽  
S A Oglesby ◽  
T K Harden
Keyword(s):  
Membrane Potential ◽  
Single Channel ◽  
Quantitative Measure ◽  
Ionophore A23187 ◽  
Intracellular Ca ◽  
Excised Patches ◽  
Patch Configuration ◽  
Single Channel Currents ◽  
Calcium Activated Potassium Channel ◽  
Channel Currents

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.

Number of KCa Channels Underlying Spontaneous Miniature Outward Currents (SMOCs) in Mudpuppy Cardiac Neurons

2001 ◽  
Vol 85 (1) ◽  
pp. 54-60 ◽  
Author(s):  
Fabiana S. Scornik ◽  
Laura A. Merriam ◽  
Rodney L. Parsons
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Current Amplitude ◽  
Channel Current ◽  
Single Channel Current ◽  
Outward Currents ◽  
Intracellular Ca ◽  
Single Channel Currents ◽  
Channel Currents

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca2+-dependent K+(BK) channels. Previously we reported that SMOCs are activated by Ca2+-induced Ca2+ release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca2+ stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K+ concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca2+, external TEA, and caffeine. In cell-attached patch recordings, 166 μM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than −20 mV, suggesting that the transient rise in intracellular Ca2+from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 μM.

A Calcium-Activated Nonspecific Cation Channel from Olfactory Receptor Neurones of the Silkmoth Antheraea Polyphemus

1991 ◽  
Vol 161 (1) ◽  
pp. 455-468
Author(s):  
F. ZUFALL ◽  
H. HATT ◽  
T. A. KEIL
Keyword(s):  
Olfactory Receptor ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Cation Channel ◽  
Voltage Response ◽  
Antheraea Polyphemus ◽  
Cell Excitability ◽  
Olfactory Receptor Neurones ◽  
Single Channel Currents ◽  
Channel Currents

Single-channel patch-clamp techniques were used to identify and characterize a Ca2+-activated nonspecific cation channel (CAN channel) on insect olfactory receptor neurones (ORNs) from antennae of male Antheraea polyphemus. The CAN channel was found both in acutely isolated ORNs from developing pupae and in membrane vesicles from mature ORNs that presumably originated from inner dendritic segments. Amplitude histograms of the CAN single-channel currents presented well-defined peaks corresponding to at least four channel substates each having a conductance of about 16 pS. Simultaneous gating of the substates was achieved by intracellular Ca2+ with an EC50 value of about 80 nmoll−1. Activity of the CAN channel could be blocked by application of amiloride (IC50 <100nmoll−1). Moreover, in the presence of 1μmoll−1 Ca2+, opening of the CAN channel was totally suppressed by 10 μmoll−1 cyclic GMP, whereas ATP (1 mmol l−1) was without effect. We suggest that the CAN channel plays a specific role in modulation of cell excitability and in shaping the voltage response of ORNs.

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