A-LTX receptors

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... 

Therefore, a-LTX receptors, which are so crucial for the toxin s action in all biological systems, have been comprehensively studied. 

The first a-LTX receptor to be identified was isolated from solubilized bovine brain by toxin-affinity chromatography in the presence of Ca2+ (Petrenko et al. 1990) and termed neurexin (NRX) (Ushkaryov et al. 1992) (Figure 3). 

In the search for a Ca2+ independent a-LTX receptor, affinity chromatography in the absence of Ca2+ was used to isolate brain proteins with any affinity for a-LTX. Sequencing of all proteins in the a-LTX column eluate (Krasnoperov et al. 1996 Krasnoperov et al. 2002b) has revealed that, in addition to LPH1, PTPg can also bind to the column as a set of two minor bands. 

The genes for all three a-LTX receptors have been knocked out in mice, and these mutations are not lethal. NRX la knockout mice show no obvious behavioral phenotype (Geppert et al. 1998). LPH1 knockout causes social problems in mice (Tobaben et al. 2002), consistent with LPH1 involvement in schizophrenia (Chen and Chen 2005). PTPo knockout leads to developmental abnormalities in the nervous system and outside it (Meathrel et al. 2002 Batt et al. 2002). 

The ability of a-LTX to trigger neurotransmitter exocytosis in the absence of extracellular Ca2+ remains particularly interesting and inexplicable to the field (Longenecker et al. 1970 Ceccarelli et al. 1979 see also Siidhof (2001) and Ushkaryov et al. (2004) for review). This is clearly different from depolarization-induced exocytosis, which is Ca2+-dependent, but not unlike the effect of hypertonic sucrose. The possibility that a-LTX-induced release involves an unknown, Ca2+-independent mechanism which may also occur during normal synaptic activity has provided the casus belli for many a quest for a-LTX structure and receptors that could trigger neurotransmission via intracellular mechanisms. 

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). 

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. 

In addition to the presence of receptors, the ionic composition of buffer (Finkelstein et al. 1976) and the makeup of lipid bilayer (Robello et al. 1984) can influence the rate of a-LTX insertion into lipid membranes. It is possible that the membrane microenvironment affects the insertion of a receptor-bound tetramer also in a physiological context. For example, at 0° C, a-LTX binds to receptor-containing membranes, but it is not inserted (Khvotchev et al. 2000 Volynski et al. 2000). 

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). 

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. 

Because NRX la requires Ca2+ to bind a-LTX, it cannot mediate the toxin s effects in the absence of Ca2+. The quest for a Ca2+-independent receptor continued, and a major protein was eventually isolated by a-LTX-chromatography... 

All a-LTX actions in biological systems require receptors, which provide binding sites for the toxin on the cell surface. Once the toxin is bound, part of its Ca2+-dependent action is due to pore formation and influx of Ca2+. This mechanism... 

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. 

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