**4.1 Patch clamp recordings**

46 Neuroscience – Dealing with Frontiers

These large channels are distinguished by their proteins which can be classified in accordance with their function, mechanism for membrane penetration, the size of their pores, its pore-forming subunits, and type of pore-forming proteins within the membrane plane (α- or β). By comparing the protein configuration in the plasma membrane it is possible to actually evolutionarily delineate the pore-forming protein of distinct organisms. Proteins that form large conductance channels in the plasma membrane have been described in virus (Madan et al, 2007), bacteria (Huffman et al, 2004), fungi (Ojcius, D. M. & Young, J. D., 1991) and plantae (Klüsener & Weiler, 1999). Mostly due to these proteins being functionally linked to the mechanism of defense (plantae) or the invasion of the hosts (virus, bacteria, etc). These proteins are often secreted into the extracellular environment as

In humans and other mammals the secreted proteins, there have been found the antimicrobial peptide families of defensins (Kagan et al 1990) and cathelicidins (Scocchi et al, 1999; Skerlavaj et al, 2001). Other peptides, such as dermicidin (Boman et al 1993; Christensen et al, 1988) and anionic peptides (Brogden et al, 1996,1997,2003) are also found in tissue-specific areas. In the immune system, these pores cause physical damage to invading pathogens. In general, pore-forming proteins are monomeric molecules that interact with each other while they are inserted into membranes, where they continue to polymerize further to form large transmembrane pores, leading to a process of antimicrobial

Generally, in vertebrates, more specifically in mammals, these pores are integral membrane proteins that are capable of opening under physiological or pathological condition. They can be found in several parts of the organism and may be associated with organism defense (release of pro-inflammatory agents, destruction of pathogens, cell death) or not (release of

The main large conductance ion channels which are found in mammals, are the connexin hemichannels (Cx43, Cx32 and other), pannexins (pannexin-1), maxi anion channel, voltagedependent anionic channel (VDAC), maxi-K channel, maitotoxin pore, transient receptor potential vanilloid type-1 (TRPV1), transient receptor potential ankhirin type-1 (TRPA1) and ATP-activated P2X pores (P2X2, P2X4 and P2X7 receptors). All of these permit the passage of mono and divalent ions and the flow of molecules of up to 1000 Da. The majority of these large conductance ion channels are preferentially permeable to cations, while VDAC and maxi anion are permeable to anions. In addition, they are all permeable to the anionic ATP4 molecule (Nagasawa et al, 2009; Pellegatti et al, 2005; Yip et al, 2009) and glutamate (Léon et

Activation of the purinergic P2X7 receptor (P2X7R) is rather unusual among ion channels. Brief agonist stimulation induces a non-selective cation-dependent pore, permitting the permeation of monovalent and divalent cations that leads to plasma membrane depolarization (Virginio et al, 1999b). By contrast, a prolonged and repetitive agonist application (at concentrations greater than 100 µM) promotes increased membrane

monomers which then oligomerize and form the channel in the host membrane.

**2. Proteins that form large channels in mammal cells** 

activity and cytotoxicity.

al, 2008).

neurotransmissor, proliferation etc).

**3. P2X7 receptor associated pore** 

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 patch-clamp technique, as depicted in Figure 2.
