**5. Electrophysiological studies of P2X7**

The first group which published a unitary current associated with the P2X7 pore was Coutinho-Silva and coworkers in 1997. They used a cell attached configuration in mouse macrophages to show that the P2X7 receptor when activated by ATP (in milimolar concentrations), is able to activate a large conductance channel with unitary conductance of about 400 pS (Coutinho-Silva et al, 1997). These pores were voltage dependent and had properties similar to P2Z permeabilization, such as uptake of large cations (N-methyl-Dglucamine) and anions (glutamate). Their opening is favored at temperatures higher than 30 degree Celsius and is blocked by oxidized ATP and Mg2+. These authors did not record this conductance in excised patches which was the first clue for the participation of secondary messengers and cytoskeletal proteins.

After this discovery, other groups used cell attached configuration however only in low currents (~8pS) was the P2X7 receptor recorded (Jiang et al, 2005; Ugur et al, 1997; Virginio et al, 1997). The large conductance activity was detected in dye uptake assays (Jiang et al, 2005; Hibell et al, 2000; Virginio et al, 1997;) or in whole cell experiments. In the whole cell, the macroscopic current induced by ATP or BzATP (a synthetic analog more potent than ATP) could be divided, according to some reports, into two distinct conductance (or components). The first component are the small channels opening and the second component is related to the large conductance of the P2X7 receptor (Jiang et al, 2005; Virginio et al, 1999).

In 2005, our group, using cell attached configuration, recorded a unitary conductance of approximately 400 pS in murine macrophages and 2BH4 cells (Faria et al, 2005). We observed a linear response in positive and negative holding potential however this large conductance channel was never observed in excised patch, supporting that there is a dependence of intracellular signals as previously suggested by Coutinho-Silva's studies (Faria et al, 2005). In addition it was found that blockers of the P2X7 receptor reduced the conductance and were modulated by the intracellular Ca2+ and MAPK. On the otherhand, Riedel's group in 2007, using the patch-clamp technique, studied the influence of external alkali and organic monovalent cations on the single-channel properties of the human P2X7 receptor. In cell attached and excised patches, they observed activity only of the small conductance channel. Interestingly, they also reported an increased probability of P2X7 small channel opening when extracellular Na+ was substituted for other monovalent cations (Riedel et al, 2007a). This same group used human P2X7 receptors expressed in *Xenopus laevis* oocytes and recorded single channels using the patch-clamp technique in the outsideout configuration. The result observed was similar to the previous paper as the large conductance channel was not observed (Riedel et al 2007b).

In 2008, Schachter and coworkers compared the P2X7 receptor pore formation of macrophages and HEK-293 cells transfected with P2X7 (HEK-P2X(7) ) receptors using patch-

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),

The first group which published a unitary current associated with the P2X7 pore was Coutinho-Silva and coworkers in 1997. They used a cell attached configuration in mouse macrophages to show that the P2X7 receptor when activated by ATP (in milimolar concentrations), is able to activate a large conductance channel with unitary conductance of about 400 pS (Coutinho-Silva et al, 1997). These pores were voltage dependent and had properties similar to P2Z permeabilization, such as uptake of large cations (N-methyl-Dglucamine) and anions (glutamate). Their opening is favored at temperatures higher than 30 degree Celsius and is blocked by oxidized ATP and Mg2+. These authors did not record this conductance in excised patches which was the first clue for the participation of secondary

After this discovery, other groups used cell attached configuration however only in low currents (~8pS) was the P2X7 receptor recorded (Jiang et al, 2005; Ugur et al, 1997; Virginio et al, 1997). The large conductance activity was detected in dye uptake assays (Jiang et al, 2005; Hibell et al, 2000; Virginio et al, 1997;) or in whole cell experiments. In the whole cell, the macroscopic current induced by ATP or BzATP (a synthetic analog more potent than ATP) could be divided, according to some reports, into two distinct conductance (or components). The first component are the small channels opening and the second component is related to the large conductance of the P2X7 receptor (Jiang et al, 2005;

In 2005, our group, using cell attached configuration, recorded a unitary conductance of approximately 400 pS in murine macrophages and 2BH4 cells (Faria et al, 2005). We observed a linear response in positive and negative holding potential however this large conductance channel was never observed in excised patch, supporting that there is a dependence of intracellular signals as previously suggested by Coutinho-Silva's studies (Faria et al, 2005). In addition it was found that blockers of the P2X7 receptor reduced the conductance and were modulated by the intracellular Ca2+ and MAPK. On the otherhand, Riedel's group in 2007, using the patch-clamp technique, studied the influence of external alkali and organic monovalent cations on the single-channel properties of the human P2X7 receptor. In cell attached and excised patches, they observed activity only of the small conductance channel. Interestingly, they also reported an increased probability of P2X7 small channel opening when extracellular Na+ was substituted for other monovalent cations (Riedel et al, 2007a). This same group used human P2X7 receptors expressed in *Xenopus laevis* oocytes and recorded single channels using the patch-clamp technique in the outsideout configuration. The result observed was similar to the previous paper as the large

In 2008, Schachter and coworkers compared the P2X7 receptor pore formation of macrophages and HEK-293 cells transfected with P2X7 (HEK-P2X(7) ) receptors using patch-

uptake of substances (fluorescent dyes) and enzymatic activity.

conductance channel was not observed (Riedel et al 2007b).

**5. Electrophysiological studies of P2X7** 

messengers and cytoskeletal proteins.

Virginio et al, 1999).

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

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

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