**4.2 Cell permeabilization assays**

This technique allows the detection of plasma membrane permeabilization in different cell types, by inducing the activation and opening of large conductance channels. In general, we use low molecular weight fluorescent dyes, such as fluorescein, lucifer yellow, calcein, ethidium bromide, propidium iodide, in order to observe the entrance of these impermeable dyes after the permeabilization phenomenon.

P2X7 receptor pore-formation has been studied by several groups in different cell types. Most experiments associated with pore formation have centered around dye uptake experiments mediated by the P2X7 receptor.

#### **4.3 Flow cytometry assays**

This is a technique for counting and examining microscopic particles, such as cells and chromosomes by suspending them in a stream of fluid and passing them through an

The Mystery of P2X7 Ionotropic Receptor:

membrane surface.

From a Small Conductance Channel to a Large Conductance Channel 49

Fig. 2. Illustration of the different modes and configurations of the patch-clamp technique. In the cell-attached mode, the patch electrode remains sealed to the cell membrane, permitting the recording of currents through single-ion channels from the patch of membrane surrounded by the tip of the electrode. This configuration can be used for

studying the activity of ion channels present in the membrane patch. In the whole cell mode, in the initial cell-attached configuration, additional suction is applied to rupture the cell membrane, thus providing access to the intracellular space. The larger opening at the tip of the patch electrode, compared with the sharp microelectrode, provides lower resistance and better electrical access to the cell- because the volume of the patch electrode is much bigger than the cell, cellular soluble contents will slowly be replaced by contents of the electrode, referred to as "dialyzing" the cell contents. Whole-cell mode records currents through all channels from the entire cell membrane at once. In the Inside-out patch mode, after the gigaseal are formed, the micropipette is quickly withdrawn from the cell, pulling off a patch of membrane from the cell, leaving the membrane patch attached to the micropipette, and exposing the intracellular surface of the membrane to the external media. This is useful when an experimenter wishes to pharmacologically manipulate the intracellular side of the ion channels. In the outside-out mode, after the whole-cell patch is formed, the electrode can be slowly withdrawn from the cell, allowing a bulb of membrane to bleb out from the cell. After the electrode to be pulled far enough away, this bleb will detach from the cell and reform as a convex membrane at the electrode end (like a ball open at the electrode tip), with the original outside of the membrane facing outward from the electrode. Single channel recordings are possible in this form if the membrane bleb is small enough. Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell, and exposed to different solutions on the extracellular

Fig. 1. Single channel and macroscopic currents in mouse peritoneal macrophages. The ionic currents were recorded at 37ºC and holding potential of -60mV using the cell attached configuration. A1- Single channel activity recorded after stimulation with 1mM ATP in the bath. A2- Single channel activity recorded after stimulation with 10uM Ionomycin in the bath. A3- Macroscopic current recorded after stimulation with 1mM ATP in the bath. A4- Macroscopic current recorded after stimulation with 10uM ioniomycin in the bath. Arrows in figure B represent the moment of application of the agonists. Under each recording there is a schematic representing the electrophysiological configuration, the localization and the agonist concentration used. A- Intracellular saline: 150mM KCl, 5mM NaCl, 0.1mM EGTA, 10mM HEPES, pH 7.4. B- Extracellular saline: 150mM NaCl, 5mM NaCl, 1mM MgCl2+, 1mM CaCl2+, 10mM Hepes, pH 7.4.

electron detection apparatus. It allows the simultaneous multiparametric analysis of the physical and chemical characteristics of thousands of particles per second. Clinical and research laboratories uses flow cytometry , to analyze multiple cell parameters such as cell cycle, cell membrane alterations and cell phenotype. Several studies have used flow cytometry in dye uptake assays stimulating the pore forming protein to open and permit the entry of fluorescent dyes.

#### **4.4 Colorimetric assays**

The measurement most commonly used to detect effects of P2X7 activation is colorimetric assays which are based on the absorbance of light. In according to Beer's Law, a solute absorbs light of a particular wavelength, and the absorbance is directly proportional to the substance concentration in solution. The measurement is done by a spectrophotometer, displaying and recording absorbance in quantifiable units. In general, the substance of

Fig. 1. Single channel and macroscopic currents in mouse peritoneal macrophages. The ionic currents were recorded at 37ºC and holding potential of -60mV using the cell attached configuration. A1- Single channel activity recorded after stimulation with 1mM ATP in the bath. A2- Single channel activity recorded after stimulation with 10uM Ionomycin in the bath. A3- Macroscopic current recorded after stimulation with 1mM ATP in the bath. A4- Macroscopic current recorded after stimulation with 10uM ioniomycin in the bath. Arrows in figure B represent the moment of application of the agonists. Under each recording there is a schematic representing the electrophysiological configuration, the localization and the agonist concentration used. A- Intracellular saline: 150mM KCl, 5mM NaCl, 0.1mM EGTA, 10mM HEPES, pH 7.4. B- Extracellular saline: 150mM NaCl, 5mM NaCl, 1mM MgCl2+,

electron detection apparatus. It allows the simultaneous multiparametric analysis of the physical and chemical characteristics of thousands of particles per second. Clinical and research laboratories uses flow cytometry , to analyze multiple cell parameters such as cell cycle, cell membrane alterations and cell phenotype. Several studies have used flow cytometry in dye uptake assays stimulating the pore forming protein to open and permit the

The measurement most commonly used to detect effects of P2X7 activation is colorimetric assays which are based on the absorbance of light. In according to Beer's Law, a solute absorbs light of a particular wavelength, and the absorbance is directly proportional to the substance concentration in solution. The measurement is done by a spectrophotometer, displaying and recording absorbance in quantifiable units. In general, the substance of

1mM CaCl2+, 10mM Hepes, pH 7.4.

entry of fluorescent dyes.

**4.4 Colorimetric assays** 

Fig. 2. Illustration of the different modes and configurations of the patch-clamp technique. In the cell-attached mode, the patch electrode remains sealed to the cell membrane, permitting the recording of currents through single-ion channels from the patch of membrane surrounded by the tip of the electrode. This configuration can be used for studying the activity of ion channels present in the membrane patch. In the whole cell mode, in the initial cell-attached configuration, additional suction is applied to rupture the cell membrane, thus providing access to the intracellular space. The larger opening at the tip of the patch electrode, compared with the sharp microelectrode, provides lower resistance and better electrical access to the cell- because the volume of the patch electrode is much bigger than the cell, cellular soluble contents will slowly be replaced by contents of the electrode, referred to as "dialyzing" the cell contents. Whole-cell mode records currents through all channels from the entire cell membrane at once. In the Inside-out patch mode, after the gigaseal are formed, the micropipette is quickly withdrawn from the cell, pulling off a patch of membrane from the cell, leaving the membrane patch attached to the micropipette, and exposing the intracellular surface of the membrane to the external media. This is useful when an experimenter wishes to pharmacologically manipulate the intracellular side of the ion channels. In the outside-out mode, after the whole-cell patch is formed, the electrode can be slowly withdrawn from the cell, allowing a bulb of membrane to bleb out from the cell. After the electrode to be pulled far enough away, this bleb will detach from the cell and reform as a convex membrane at the electrode end (like a ball open at the electrode tip), with the original outside of the membrane facing outward from the electrode. Single channel recordings are possible in this form if the membrane bleb is small enough. Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell, and exposed to different solutions on the extracellular membrane surface.

The Mystery of P2X7 Ionotropic Receptor:

cationic pathway were unidentified (Schachter et al, 2008).

and the large conductance channel not recorded (Flittiger et al, 2010).

conductance (pore), while in rats there was dependency (Roger et al, 2010)

**6. The search of large channels associated with P2X7**

From a Small Conductance Channel to a Large Conductance Channel 51

clamp recordings. They did not record a unitary conductance activity in transfected HEK-293 cells. A pore with conductance of approximately 400 pS was recorded only in mouse peritoneal macrophages. Using dye uptake experiments, they also observed the differential uptake of cations and anions between the endogenous P2X7 receptor pore formation in macrophages. The anionic pathways associated with the large conductance channel and the

In 2009, based on previous data reporting that increasing intracellular Ca2+ is able to induce the opening of a pore biophysically similar to the P2X7 receptor pore. Investigating this channel we found that calcium ionophores at micromolar concentrations induced dye uptake and ionic currents presented a unitary conductance of 400 pS, in the attached cell. This pore was unaffected by P2X7 receptor blockers, but it had intracellular signaling components similar to the P2X7 receptor large conductance channel and not observed in excised patches. However, we did not identify the entity responsible for inducing intracellular Ca2+ pore opening in mouse macrophages and 2BH4 cells (Faria et al, 2009).

In 2010, Yan and collaborators performed experiments to understand how the three binding site occupation for the ATP may affect the P2X7 receptor gating. They showed that ATP concentrations in the milimolar range were able to biphasically activate and deactivate native receptors while micromolar concentrations responded monophasically. Both phases of response were abolished by the application of Az10606120, a P2X7R-specific antagonist. This slow secondary growth of current in the biphasic response coincided temporally with pore dilation. This pore current was insensitive to Na+ and Ca2+ influx and fully reestablished the initial gating properties after 30 min of washout. The complex pattern of gating exhibited by wild-type channels can be accounted for by the Markov state model that includes the negative cooperativity of agonist binding to unsensitized receptors caused by the occupancy of one or two binding sites, thus opening the channel pore to a low conductance state when the two sites are bound, and when three sites are occupied, triggering a high conductance state (pore dilation) (Yan et al, 2010). In contrast, Flittiger and collaborators while investigating the participation of protons in the activation of hP2X7Rs observed that human P2X7 receptors expressed in *Xenopus laevis* oocytes were activated by ATP or BzATP at different pH values. The unitary currents were blocked by protonation

In 2010, Roger and coworkers, used a whole cell configuration to characterize the functional properties of the biphasic ionic conductance recorded in human and rat P2X7 receptors. They observed that in humans there was a Ca2+/calmodulin independence of the secondary

In search of the P2X7 receptor protein responsible for large unitary conductance and the pore that is induced rising intracellular Ca2+ (Faria et al, 2009) we compared the main biophysical properties of the other large conductance channels with these types of pores.

In cultured cells under resting conditions, hemichannels have a low open probability at negative membrane potentials, and the open probability is increased at positive potentials (Bukauskas & Verselis, 2004). Increases in hemichannel levels have been clearly associated

interest, by itself, does not absorb light in the wavelength used. We have to apply one or more reagents to produce colored compounds which are proportional to the substance concentration of the unknown. These methodologies may be used to measure cell death (LDH release, MTT assay), release of substances (neurotransmissors, cytokines and others), uptake of substances (fluorescent dyes) and enzymatic activity.
