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

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

The Mystery of P2X7 Ionotropic Receptor:

probability at both single-channel and whole-cell levels.

second stage of conductance of the TRPV1 channel.

experimental conditions.

(Gunthorpe et al, 2000).

**7. Conclusion** 

From a Small Conductance Channel to a Large Conductance Channel 53

exhibit a time dependent inactivation at large positive and negative potentials over ±15 to +30 mV. The voltage dependence of open probability (Popen) remains bell-shaped with maximum at a voltage near 0 mV. These results indicate that the channel is highly selective to anions (Mitchell et al, 1997; Schlichter et al, 1990) but that the degree of anion selectivity may vary (Bajnath et al, 1993, Kemp et al, 1993) not only with cell types but also with the

TRPV1 is a nonselective cation channel which is structurally related to the voltage-activated potassium (Kv) channels. The TRPV1 expressed alone in human embryonic kidney-derived HEK293 cells or *Xenopus* oocytes can account for the majority of the electrophysiological properties exhibited by native capsaicin receptors in sensory neurons, including ligand affinity, permeability sequence, current/voltage (I/V) relationship, conductance and open

In whole cell configuration, I/V relationships have reversal potentials close to 0 mV, indicating the opening of non-selective cationic channels, and a substantial outwards rectification with a region of negative slope conductance at potentials negative to +70 mV

In cell attached configuration, the single-channel amplitude histogram showed two well separated peaks representing open and closed states, without sub-conductance levels observed at +60 mV. The single-channel amplitude at +60 mV was 5.3 pA, corresponding to a conductance of 88.3 pS. Open probability depends on membrane potential as it has been shown in single-channel and whole-cell recordings (Premkumar *et al.* 2002; Voets *et al.*  2004b). At the molecular level, an extracellular Ca2+-dependent reduction of TRPV1 responsiveness upon continuous vanilloid exposure (electrophysiological desensitization) may underlie this phenomenon, at least in part (Caterina et al, 1997; Szallasi & Blumberg, 1999). In relation to TRPV1 large conductance formation, some papers have published that this channel may mediate fluorescent dye uptake, however they did not characterize the biophysical properties related to this phenomenon (Blumberg, 2007; Hellwig et al, 2004; Myrdal & Steeiger, 2005). Since 2008, other research groups have now characterized this

MTX may initially induce the activation of a NSCC, which is permeable to Na+ and K+, but it has a low permeability to Ca2+ (Schilling et al., 1999; de la Rosa et al., 2007). MTX may also increase [Ca2+]i via various Ca2+ entry pathways following depolarization, including Ltype voltage-sensitive Ca2+ channels (VSCCs), which are the predominant Ca2+ channel type in vascular smooth muscle (Sanders, 2001). The unitary conductance was 12 pS in the presence of 50 mM Ba2+. Within a burst, the distribution of opening times was a single exponential with a mean open time of 10.4 ms (Kobayashi et al, 1987-Br J Pharmacol). GH4C1 rat pituitary cells were stimulated with independent currents of an MTX-induced steady-state voltage of nearly 400 pS/pF within seconds of addition to the bath. Ion substitution experiments demonstrated that these ionic currents are consistent with the

In summary, Maxi anion and pl-VDAC had unitary conductance of 400 pS, but were voltage-dependent and anionic. In relation to Maitotoxin and TRPV1 pores which were not

conductance of sodium and chloride, but not calcium ions (Young et al, 1995).

with rises in intracellular-free Ca2+ concentration ([Ca2+]i) (Schalper et al, 2008, Sánchez et al, 2009). Normally, positive membrane potentials activate the fast gating, which corresponds to fast transitions between the fully open state and a substate. At negative membrane potentials, the loop gating activates slow transitions, perhaps involving multiple substates between the fully open state, substates and the fully closed state (Bukauskas & Verselis, 2004). Unitary conductance of the connexin hemichannel recorded in a cell attached configuration exhibited conductance values of approximately 300 pS for connexin 56 (Ebihara et al, 1999), for connexin 43 approximately of 200 pS (Contreras et al, 2003a, 2003b; Kang et al, 2008; Retamal et al, 2007b), 250 pS for connexin 46 (Ma & Dahl, 2006) and 200 pS for connexin 50 hemichannel (Liu et al, 2011).

Panx1 channels have been reported with different voltage dependence. In some cases, Panx1-mediated currents are outwardly rectifying and require a depolarization for activation (R. Bruzzone et al, 2003, Pelegrin et al, 2006). This observation is at odds with currents recorded in pyramidal neurons following Panx1 activation by ischemia or NMDAR stimulatio, where the current-voltage (I-V) relationship is clearly linear (Thompson et al, 2006, 2008a, 2008b) similar to Panx1 when it is conducting ATP (Bao et al, 2004). Upon activation, pannexons open into large non-selective pores, which are insensitive to physiological levels of extracellular Ca2+ but they are permeable to ions and small molecules as well as metabolites of up to 1000 Da and a wide range of membrane depolarization levels (S. Bruzzone et al, 2004). In addition, some groups have measured the unitary conductance of this large conductance channel, which is approximately 500 pS (Bao et al, 2004; Locovei et al, 2006; Thompson et al, 2006) which suggests that depolarization activates rectifying Panx1 currents and that other mechanisms lead to currents that are not significantly rectifying.

The presence of VDAC in the plasma membrane (pl-VDAC) would be expected to be lethal to the cell (Yu and Forte, 1996). However, considering the resting membrane potential across the plasma membrane of about -30 to -60 mV (Dermietzel et al, 1994), VDAC1 in the plasma membrane would be in a closed state most of the time (Mannella, 1997). Current events were recorded from excised patches of plasma membranes of a rat astrocytic cell line (RGCN)where it was found that the underlying channels exhibited a conductance from 401 to 250 pS. Open probability was the highest between 210 mV, and gradually approached zero beyond 225 mV. Activity induced by voltage ramps between 240 mV appeared after a several minute delay.

Several authors have reported the single-channel opening of the Maxi-anion with larger unitary conductance (300–400 pS) recorded in the cell-attached mode after cell swelling (Dutta et al, 2004; Liu et al, 2006, 2008a). Most authors noted that the maxi-anion channel has multiple subconductance states of various levels, such as 15, 50, 100, 150 and 200 pS (Dutta et al, 2004; Olesen & Bundgaard, 1992; Schwarze & Kolb, 1984; Akanda et al, 2008). When the extracellular Cl- concentration varied, the single-channel conductance saturated at 640 pS with Km = 112 mM in L6 myoblasts (Hurnák & Zachar, 1994), at 581 pS with Km = 120 mM in T lymphocytes (Schlichter et al, 1990) and at 617 pS with Km = 77 mM in frog skeletal muscle ''sarcoballs'' (Hals et al, 1989). The maxi-anion channel presents roughly uniform behavior in different cell types. The current–voltage relationship of the fully open state is usually symmetrical and linear with no rectification when it is recorded by cell attached configuration. The channel has a maximal open channel probability at around 0 mV, but it readily closes when the voltage exceeds a range of ±15 to ± 30 mV. The macroscopic currents

with rises in intracellular-free Ca2+ concentration ([Ca2+]i) (Schalper et al, 2008, Sánchez et al, 2009). Normally, positive membrane potentials activate the fast gating, which corresponds to fast transitions between the fully open state and a substate. At negative membrane potentials, the loop gating activates slow transitions, perhaps involving multiple substates between the fully open state, substates and the fully closed state (Bukauskas & Verselis, 2004). Unitary conductance of the connexin hemichannel recorded in a cell attached configuration exhibited conductance values of approximately 300 pS for connexin 56 (Ebihara et al, 1999), for connexin 43 approximately of 200 pS (Contreras et al, 2003a, 2003b; Kang et al, 2008; Retamal et al, 2007b), 250 pS for connexin 46 (Ma & Dahl, 2006) and 200 pS

Panx1 channels have been reported with different voltage dependence. In some cases, Panx1-mediated currents are outwardly rectifying and require a depolarization for activation (R. Bruzzone et al, 2003, Pelegrin et al, 2006). This observation is at odds with currents recorded in pyramidal neurons following Panx1 activation by ischemia or NMDAR stimulatio, where the current-voltage (I-V) relationship is clearly linear (Thompson et al, 2006, 2008a, 2008b) similar to Panx1 when it is conducting ATP (Bao et al, 2004). Upon activation, pannexons open into large non-selective pores, which are insensitive to physiological levels of extracellular Ca2+ but they are permeable to ions and small molecules as well as metabolites of up to 1000 Da and a wide range of membrane depolarization levels (S. Bruzzone et al, 2004). In addition, some groups have measured the unitary conductance of this large conductance channel, which is approximately 500 pS (Bao et al, 2004; Locovei et al, 2006; Thompson et al, 2006) which suggests that depolarization activates rectifying Panx1 currents and that other mechanisms lead to currents that are not significantly rectifying.

The presence of VDAC in the plasma membrane (pl-VDAC) would be expected to be lethal to the cell (Yu and Forte, 1996). However, considering the resting membrane potential across the plasma membrane of about -30 to -60 mV (Dermietzel et al, 1994), VDAC1 in the plasma membrane would be in a closed state most of the time (Mannella, 1997). Current events were recorded from excised patches of plasma membranes of a rat astrocytic cell line (RGCN)where it was found that the underlying channels exhibited a conductance from 401 to 250 pS. Open probability was the highest between 210 mV, and gradually approached zero beyond 225 mV. Activity induced by voltage ramps between 240 mV appeared after a

Several authors have reported the single-channel opening of the Maxi-anion with larger unitary conductance (300–400 pS) recorded in the cell-attached mode after cell swelling (Dutta et al, 2004; Liu et al, 2006, 2008a). Most authors noted that the maxi-anion channel has multiple subconductance states of various levels, such as 15, 50, 100, 150 and 200 pS (Dutta et al, 2004; Olesen & Bundgaard, 1992; Schwarze & Kolb, 1984; Akanda et al, 2008). When the extracellular Cl- concentration varied, the single-channel conductance saturated at 640 pS with Km = 112 mM in L6 myoblasts (Hurnák & Zachar, 1994), at 581 pS with Km = 120 mM in T lymphocytes (Schlichter et al, 1990) and at 617 pS with Km = 77 mM in frog skeletal muscle ''sarcoballs'' (Hals et al, 1989). The maxi-anion channel presents roughly uniform behavior in different cell types. The current–voltage relationship of the fully open state is usually symmetrical and linear with no rectification when it is recorded by cell attached configuration. The channel has a maximal open channel probability at around 0 mV, but it readily closes when the voltage exceeds a range of ±15 to ± 30 mV. The macroscopic currents

for connexin 50 hemichannel (Liu et al, 2011).

several minute delay.

exhibit a time dependent inactivation at large positive and negative potentials over ±15 to +30 mV. The voltage dependence of open probability (Popen) remains bell-shaped with maximum at a voltage near 0 mV. These results indicate that the channel is highly selective to anions (Mitchell et al, 1997; Schlichter et al, 1990) but that the degree of anion selectivity may vary (Bajnath et al, 1993, Kemp et al, 1993) not only with cell types but also with the experimental conditions.

TRPV1 is a nonselective cation channel which is structurally related to the voltage-activated potassium (Kv) channels. The TRPV1 expressed alone in human embryonic kidney-derived HEK293 cells or *Xenopus* oocytes can account for the majority of the electrophysiological properties exhibited by native capsaicin receptors in sensory neurons, including ligand affinity, permeability sequence, current/voltage (I/V) relationship, conductance and open probability at both single-channel and whole-cell levels.

In whole cell configuration, I/V relationships have reversal potentials close to 0 mV, indicating the opening of non-selective cationic channels, and a substantial outwards rectification with a region of negative slope conductance at potentials negative to +70 mV (Gunthorpe et al, 2000).

In cell attached configuration, the single-channel amplitude histogram showed two well separated peaks representing open and closed states, without sub-conductance levels observed at +60 mV. The single-channel amplitude at +60 mV was 5.3 pA, corresponding to a conductance of 88.3 pS. Open probability depends on membrane potential as it has been shown in single-channel and whole-cell recordings (Premkumar *et al.* 2002; Voets *et al.*  2004b). At the molecular level, an extracellular Ca2+-dependent reduction of TRPV1 responsiveness upon continuous vanilloid exposure (electrophysiological desensitization) may underlie this phenomenon, at least in part (Caterina et al, 1997; Szallasi & Blumberg, 1999). In relation to TRPV1 large conductance formation, some papers have published that this channel may mediate fluorescent dye uptake, however they did not characterize the biophysical properties related to this phenomenon (Blumberg, 2007; Hellwig et al, 2004; Myrdal & Steeiger, 2005). Since 2008, other research groups have now characterized this second stage of conductance of the TRPV1 channel.

MTX may initially induce the activation of a NSCC, which is permeable to Na+ and K+, but it has a low permeability to Ca2+ (Schilling et al., 1999; de la Rosa et al., 2007). MTX may also increase [Ca2+]i via various Ca2+ entry pathways following depolarization, including Ltype voltage-sensitive Ca2+ channels (VSCCs), which are the predominant Ca2+ channel type in vascular smooth muscle (Sanders, 2001). The unitary conductance was 12 pS in the presence of 50 mM Ba2+. Within a burst, the distribution of opening times was a single exponential with a mean open time of 10.4 ms (Kobayashi et al, 1987-Br J Pharmacol). GH4C1 rat pituitary cells were stimulated with independent currents of an MTX-induced steady-state voltage of nearly 400 pS/pF within seconds of addition to the bath. Ion substitution experiments demonstrated that these ionic currents are consistent with the conductance of sodium and chloride, but not calcium ions (Young et al, 1995).
