**The Mystery of P2X7 Ionotropic Receptor: From a Small Conductance Channel to a Large Conductance Channel**

R.X. Faria, L.G.B. Ferreira and L.A. Alves *Laboratory of Cellular Communication, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil* 

### **1. Introduction**

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#### **1.1 Ion channels**

Ion channels are aqueous pores which allow ions to pass based upon an electrochemical gradient. In order to conduct ions, these channels modify their shape by a process called gating, a shifting between opening and closing states. These channels are gated by various stimuli i.e. applied electric field, chemical transmitters, pH, heat and cold, among others. When an electrical potential is held constant in a cell, the gating process equilibriates rather than reaching an energy-dissipating position (a cyclic steady state). Ions diffuse through open channels very fast, involving an interaction between ions, the pore, and solvent that lead to ionic selectivity, saturation, block and flux coupling (Hille, 1975a; Läuger, 1973a).

The ion channel has generally been viewed as having unchanging selectivity strengthening the argument that there is a degree of specificity between ion and channel. Nevertheless, some classes of ion channels present a more varied selectivity of ions, allowing impermeable ions or molecules to permeate under certain circumstances. However, the there is no a systematic approach to distinguish different ion channels and in most cases, it is necessary to differentiate the channels by their kinetics, molecular sequence, pharmacological properties, and response to ionic substitution.

The diversity of channels continues to pose numerous areas to explore in physiology, molecular biology, pharmacology and biophysics.(Hille, 1991). Ion channels are generally classified into two groups: the channels that allow the passage of ions (Na+, K+, Ca2+) and those that allow the passage of large ions and solutes (ie ATP, glutamate, fluorescent dyes of low molecular weight). This second group is predominantly made up of large conductance ion channels which when activated change the permeability of cell membranes for molecules of up to 1000 Daltons (Ojcius, D. M. & Ding-E Young, J., 1990; Iacovache et al, 2010). These channels are and allow for the secretion of ATP and other intracellular molecules, not to mention implicated in various mechanisms of cell death.

The Mystery of P2X7 Ionotropic Receptor:

639) or lucifer yellow (MW 457).

in plasma membrane.

pores in mammalian cells.

**4.1 Patch clamp recordings** 

patch-clamp technique, as depicted in Figure 2.

dyes after the permeabilization phenomenon.

experiments mediated by the P2X7 receptor.

**4.3 Flow cytometry assays** 

**4.2 Cell permeabilization assays** 

From a Small Conductance Channel to a Large Conductance Channel 47

permeability, allowing cellular uptake of fluorescent dyes such as propidium iodide (MW

In the last years, some research groups have provided evidence that pannexin-1 (Panx1) hemichannel might be the protein associated with P2X7 receptor pore formation. However, several cells allow the passage of dyes, despite having the pannexin-1 channel blocked (Faria et al, 2005, 2010; Schachter et al 2008; Yan et al 2008). This opens the possibility that other proteins may participate in the formation of the large channel associated with the P2X7 receptor. As mentioned above, there are other proteins capable of forming large pores

In keeping with this idea, Faria and collaborators (2009) showed that calcium ionophores may massively increase the intracellular Ca2+ and induce dye uptake. They observed a pore with biophysical and pharmacological characteristics similar to P2X7 receptor pore (Figure 1). In addition, Schilling and colleagues (1999) observed the Maitotoxin opening, a pore able to uptake fluorescent dyes. This pore was also biophysically similar to the P2X7 receptor pore. Herein, we will address some questions about the possible protein candidate (or candidates) responsible for the P2X7 receptor pore formation described above. Moreover, we will discuss the possible events which might be occurring to regulate the opening of these

In patch-clamp recording, the microelectrode is a micropipette with a relatively large tip diameter. The microelectrode is placed next to a cell and a gentle suction is applied through the microelectrode to draw a piece of the cell membrane (the 'patch') into the microelectrode tip; the glass tip forms a high resistance 'seal' with the cell membrane. The suction causes the cell to form a tight and high-resistance seal around electrode, resulting in a resistance of approximately 10 giga Ohms, which is called a gigaseal. There are several variations of the

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

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

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

**4. Current methodologies to study large conductance channels** 
