A-LTX channel

Above this, in the centre of the bowl, the four heads form a cylindrical assembly surrounding the channel (Figure lc), which is restricted at one point to 10 A (Figure2 see also Section 3.4.7). This constriction most probably corresponds to the cation binding site (selectivity filter) of the a-LTX channel (Section 3.4.1). 

Fig. 2 Membrane topography of the a-LTX pore. Cross-section of the a-LTX tetramer embedded in a membrane (as observed in liposomes) (Orlova et al. 2000) is shown alongside the cut-open voltage-dependent K+ channel (Kvl.2) (Long et al. 2005) and Ca2+ release channel (ryanodine receptor) (Serysheva et al. 2005). Fully hydrated cations and molecules known to permeate through the respective channels are shown next to each reconstruction (FITC, fluoresceine isothiocyanate NE, norepinephrine). The narrowest part of the a-LTX channel is 10 A. Molecular images were produced using the UCSF Chimera package (Pettersen et al. 2004).

The wings extend sideways from the body domains perpendicular to the central symmetry axis of the tetramer and could participate in the binding to some receptors (see Sections 2.3 and 4). They also seem to mediate homotypic interactions, causing tetramers to assemble into flat 2D crystals, often containing large numbers of tetramers (Lunev et al. 1991). These lattices could underlie the frequently described phenomenon of a-LTX channel clusterization (Robello et al. 1987 Krasilnikov and Sabirov 1992 Filippov et al. 1994 Van Renterghem et al. 2000). 

Once formed, the a-LTX channel only mediates cationic currents, probably because negatively charged acidic side chains line the channel (Finkelstein et al. 1976). Most significantly, the a-LTX channel is permeable to Ca2+ (Finkelstein et al. 1976 Krasil nikov et al. 1982 Mironov et al. 1986), and it is this aspect of the a-LTX channel that is most often considered. However, this channel is not very selective, and Ba2+, Sr2+, Mg2+ as well as Li+, Cs+ and, importantly, Na+ and K+ currents are also carried by the channel (Krasil nikov et al. 1982 Mironov et al. 1986). 

A detailed analysis of the permeability of a-LTX channels to cations and nonelectrolytes (Mironov et al. 1986 Robello 1989 Krasilnikov and Sabirov 1992) postulates that the negatively charged selectivity filter of the channel is located close to the extracellular side of the membrane, fitting in with the hypothesis that the C-terminal head domains form the selectivity filter (see Section 2.2.2). 

Although Ca2+ only carries a small proportion of currents through cell membrane-inserted a-LTX channels (Hurlbut et al. 1994 Tse and Tse 1999), the influx of Ca2+ through presynaptically-targeted a-LTX channels is most often referred to, because of the well-established link between presynaptic [Ca2+] and neurotransmitter release. There is a wealth of evidence indicating that in conditions favorable to channel formation (e.g., in the presence of divalent cations), influx of extracellular Ca2+ through a-LTX channels is an important aspect of a-LTX action. 

Such currents probably account for at least one of a-LTX actions tetrodotoxin-insensitive depolarization of artificial and biological membranes (Grasso and Senni 1979 Nicholls et al. 1982 Scheer et al. 1986). Although depolarization could cause neurotransmitter release by activating voltage-gated Ca2+ channels, Ca2+ flow through the a-LTX channel probably overwhelms this effect (Nicholls et al. 1982), so the role of depolarization in a-LTX action is unclear. 

In addition, Na+ currents through a-LTX channels can underlie some of the a-LTX-induced nonvesicular neurotransmitter release by causing, for example, the collapse of the cross-membrane Na+ gradient and the reversal of some (McMahon et al. 1990) but not all (Deri and Adam-Vizi 1993) transmitter uptake pumps. 

Several plausible hypotheses can explain this variability. Firstly, the venom contains a range of pore-forming toxins with varying conductances. Secondly, there is evidence that the make-up of the permeated membrane (Robello et al. 1984 Scheer et al. 1986 Krasilnikov and Sabirov 1992) and variations in lipid packing and order (Chanturia and Lishko 1992), but not the type of receptor present (Hlubek et al. 2000 Van Renterghem et al. 2000), can affect the properties of a-LTX channels. 

Interestingly, Ca2+ inhibits the conductance of a-LTX channels to monovalent cations, causing a flickery block, in artificial membranes (Mironov et al. 1986 Krasil nikov et al. 1988), neuroblastoma cells (Hurlbut et al. 1994), and embryonic kidney cells (Hlubek et al. 2000), although this is not apparent in receptor-expressing oocytes (Filippov et al. 1994). Mg2+ positively modulates the conductivity of toxin channel for Ca2+ (Davletov et al. 1998 Van Renterghem et al. 2000). 

The a-LTX pore is permeable to alkaline earth cations, whose affinities for the channel decrease in the following sequence Mg2+ > Ca2+ > Sr2+ > Ba2+ (Mironov et al. 1986). Transition metal cations (Cd2+ > Co2+ > Ni2+ > Zn2+ > Mn2+) strongly block Ca2+ and K+ currents through a-LTX channels in artificial membranes (Mironov et al. 1986). This block is only effective when the cation is applied from the cfs-side (equivalent to the extracellular side) of the membrane. 

It is possible that the water-filled a-LTX channel, which is relatively wide ( 10A at its narrowest (Krasilnikov and Sabirov 1992 Orlova et al. 2000), can pass small molecules. Indeed, a-LTX channels inserted in the membranes of synaptosomes, NMJ nerve terminals, and receptor-transfected COS7 cells appear to pass fluorescein (Stokes-Einstein radius, Re = 4.5 A) and norepinephrine (Re < 4 A) (Davletov et al. 1998 Rahman et al. 1999 Volynski et al. 2000), shown in Figure 2 for comparison with 8-hydrated calcium ion (Rc = 4.2 A) and the toxin channel. Analysis of impermeant cations commonly used in channel studies reveals that a-LTX channels are poorly permeable (Hurlbut et al. 1994) to glucosamine H+(Re = 4.6 A) and not significantly permeable (Tse and Tse 1999) to N-methyl-D-glucamine (Re = 5.2 A), thus limiting the pore diameter by 10 A. 

These observations are particularly relevant to the experimental use of a-LTX in neurotransmission studies, since a-LTX has been shown in several systems to cause nonvesicular release by allowing leakage of cytoplasmic neurotransmitters (McMahon et al. 1990 Deri et al. 1993 Davletov et al. 1998). This flux could be mediated by the a-LTX channel itself, by local disruptions of cellular membranes, or by reversal of transmitter uptake pumps driven by Na+ gradient (see Section 3.4.3). Synaptosomes seem to be particularly sensitive to an increase in hydrostatic pressure, which may occur when influx of Na+ or Ca2+ leads to a concomitant influx of water. 

It is now universally accepted that a-LTX can insert into, andpermeabilize, artificial and biological membranes. Cation currents can explain some, but not all, of the toxin s effects. For example, it is not clear how the a-LTX channel could mediate Ca2+ -independent exocytosis in neurons. Although it would be tempting to assign some of a-LTX actions in the absence of Ca2+ to Na+ currents, lack of Na+ does not prevent Ca2+-independent secretion (Tsang et al. 2000). Cation flux-associated incursion of terminals by water could be involved, but a-LTX effect on intracellular osmotic pressure has not been characterized yet. 

In the absence of Ca2+e, a-LTX only binds to LPH1 and PTPc. Ca2+-independent exocytosis requires the presence of Mg2+ and toxin insertion into the plasma membrane, but these conditions also induce formation of a-LTX channels. Influx of Na+ and efflux of K+ through these channels and associated efflux of small molecules and influx/efflux of water may cause secretion. In addition, transmitter release can be caused by membrane perturbation or direct interaction with secretory machinery. Some secretion may be nonvesicular. Receptor-mediated signaling can cause the activation of PKC in some cells. However, Ca2+-independent release is blocked by La3+, indicating that toxin pores play a crucial role in this release. 

From the earliest description of the toxin s actions on neuronal systems, it emerged that a-LTX affects specifically the presynaptic element, from which it causes massive neurotransmitter release (e.g., Longenecker et al. 1970). The toxin has no major enzymatic activities (Frontali et al. 1976). Crucially, a-LTX has been discovered to create Ca2+-permeable channels in lipid bilayers (Finkelstein et al. 1976), and a large body of evidence shows that Ca2+ influx through membrane channels induced by a-LTX in the presynaptic membrane accounts for a major part of its effect. Pore formation occurs in all the biological systems mentioned above, but the features of a-LTX-triggered release cannot be fully explained by the toxin pore. 

How the hydrophilic a-LTX inserts into lipid membranes and makes cation-permeable pores is not fully known, but an in-depth insight into the mechanisms of channel formation has been gained by combining cryo-EM, biochemical and biophysical studies with toxin mutagenesis. a-LTX pore formation consists of at least three steps toxin tetramerisation, interaction with a specific cell-surface receptor and, finally, membrane insertion. Many experimental procedures can affect some of these steps and thereby prevent or assist channel formation. 

Although a-LTX is able to insert into pure lipid membranes (Finkelstein et al. 1976), reconstituted receptors greatly enhance the rate of insertion (Scheer et al. 1986). Biological membranes seem even more refractive to the toxin when cells do not possess a-LTX receptors, no pore formation can be detected (Hlubek et al. 2000 Van Renterghem et al. 2000 Volynski et al. 2000), whereas expression of exogenous receptors allows abundant a-LTX insertion and concomitant channel... 

This hypothesis finds additional confirmation in the features of the a-LTX truncation mutants described above (Section 3.3) (Li et al. 2005). These mutants have 1 to 8 ankyrin repeats removed from their C-termini and form cation channels of dramatically different conductivities. For example, a-LTxAl mutant mediates an enormous conductance, by far exceeding that of wild-type toxin. It is possible that the removal of the last ankyrin repeat lifts an obstruction for cation movements in the extracellular mouth of the channel. In contrast, a-LTxA2 and a-LTxA3 make very inefficient channels, probably due to perturbations of the channel lining. Finally, a-LTxA8 does not induce any cation currents, as it seemingly cannot form tetramers. 

Millimolar concentrations of trivalent cations, such as Yb3+, Gd3+, Y3+, La3+ and Al3+, block the pore-mediated effects of a-LTX in synaptosomes (Scheer 1989). The nature of this effect is complex. For example, Al3+ blocks the binding of a-LTX to synaptosomes (Scheer 1989), while La3+ prevents tetramerization (Ashton et al. 2000) and thereby channel insertion and formation. 

Most trivalent cations, at 50-100pM, block previously inserted channels (Scheer 1989 Hurlbut et al. 1994 Van Renterghem et al. 2000) and inhibit a-LTX-mediated Ca2+-uptake, while La3+ blocks depolarization as well (Scheer 1989). Channel inhibition by trivalent cations is very important because La3+ is essentially the only reagent that blocks the Ca2+-independent a-LTX-evoked neurotransmitter secretion in neurons (Scheer 1989 Capogna et al. 2003). 

Fig. 6 Diverse mechanisms of a-LTX action. Right, Ca2+ is present in the medium. The pathways shown are described in the text. CC Ca2+ channels DAG, diacyl glycerol LTX 4x, a-LTX tetramers MC, mitochondria. Left, Ca2+-free conditions. For main comments, see text. The possible pathways for Ca2+-independent exocytosis shown include (1) high concentrations of Na+ mimicking Ca2+ (2) the internalised domains of a-LTX interacting with components of the exo-cytotic machinery (E) (3) a-LTX exerting direct fusogenic action.

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