Membrane channel formation

Botulinum and tetanus neurotoxins, which are extensively soluble in water, are known to form efficient membrane channels at a low pH in artificial membranes (Hoch et al. 1985 Boquet and Duflot, 1982). Membrane channel formation by a water soluble protein is an intriguing phenomenon, because for water solubility, hydrophobic domains are needed on the surface of a protein whereas for membrane channel formation, adequate hydrophobic segments will be required for the interaction with non-polar membrane bilayer. A major question to be answered is how are the polypeptides integrated in the lipid bilayer. Are the hydrophobic segments of these neurotoxins hidden in aqueous medium which get exposed... 

Buckley, J. T., 1992, Crossing three membranes. Channel formation by aerolysin. FEES Lett. 307 30-33. 

Singh, B. R., Ledoux, D. N. and Fu, F.-N., 1994, An analysis of the protein structure of botulinum and tetanus neurotoxins to understand molecular basis of membrane channel formation. In Advances in Venom and Toxin Research (Tan, N. H., Oo, S. L., Thambyrajah, V. and Azila, N.. eds.), Malaysian Society on Toxinology, Kuala Lumpur, pp. 103-108. 

In electrochemistry similar phenomena are observed, for example, with the formation of insoluble films on electrodes or with ion selective channel formation in bilayer lipid membranes or nerve cell membranes (pages 377 and 458). 

Some microbes are able to decrease the permeability of their membranes to prevent toxic metals from entering. If the toxic metals are not able to physically enter the cell, they will not be able to affect vital metal-sensitive structures, such as proteins. One way to prevent heavy metals from entering is by decreasing the production of membrane channel proteins.18 It is also possible for the metal-binding sites in the membrane and periplasm to be saturated with nontoxic metals.37 A third possibility is the formation of an extracellular polysaccharide coat, which binds and prevents metals from reaching the surface of the cell.24,38... 

The arrival of the action potential at the presynaptic terminal opens voltage-dependent Ca ion channels in the plasma membrane so that the Ca ions enter the cytosol down their concentration gradient. This results in activation of a Ca -binding cytosolic or a membrane protein. This facilitates movement of the vesicles to the membrane and formation of a fusion pore through which the neurotransmitter is discharged into the synaptic cleft (i.e. exocytosis). This occurs within about 0.1 ms of the arrival of the depolarisation (Figure 14.8). The process of exocytosis lasts for only a short time, since the Csl ion concentration in the cytosol is rapidly lowered due to the ion extrusion from the cell (Appendix 14.3). 

Amphotericin B (AmB) is a polyenic macrolide used in fungal infections and leishmaniasis, despite severe side effects (Figure 4.60). Fluorination of the macrolide skeleton has been performed using Selectfluor for F NMR studies on the mechanism of ion-channel formation in membranes by amphotericin B. ... 

In spite of the overwhelming importance of the channel mechanism for the transport of alkali and alkaline earth metal ions in biological systems, only carrier transport has been studied extensively by chemists. Studies on ion channel mimics of simple structures have long been limited to antibiotic families of gramicidin, amphotericin B, and others. Several pioneers have reported successful preparation of non-peptide artificial channels. However, their claims have been based on kinetic characteristics observed for the release of metal ions through liposomal membrane and lacked the very critical proofs of channel formation. Such a situation was... 

Figure 19. (A) Monensin modified channel forming unit 15, negatively charged a,(o-bifunctional amphiphile 16a and neutral one 16b, capable of forming monolayered membrane and positively charged bolaamphiphiles 17 as a sealing agent of the channel. (B) Model of channel formation by 15 in the monolayered membrane composed of 16 and the proposed blocking mode by 17." ...

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 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. 

Hlubek MD, Stuenkel EL, Krasnoperov VG et al (2000) Calcium-independent receptor for a-latrotoxin and neurexin la facilitate toxin-induced channel formation evidence that channel formation results from tethering of toxin to membrane. Mol Pharmacol 57 519-28 Hurlbut WP, Ceccarelli B (1979) Use of black widow spider venom to study the release of neurotransmitters. Adv Cytopharmacol 3 87-115 87-115 Hurlbut WP, Chieregatti E, Valtorta F et al (1994) a-Latrotoxin channels in neuroblastoma cells. JMembr Biol 138 91-102... 

In conclusion, it should be noted that PolyPs are polyfunctional compounds. Their most important functions are as follows phosphate and energy reservation, sequestration and storage of cations, formation of membrane channels, participation in phosphate transport, involvement in cell-envelope formation and function, gene activity control, regulation of enzyme activities, and, as a result, an important role in stress response and stationary-phase adaptation. 

Using the principle of ion pair formation between ammonium cations and the phosphate anions of lipids, Matile et al.33 prepared 8, an amphiphilic polyamine dendrimer. Rather than acting as a membrane channel, 8 was expected to form reversible membrane defects in the lipid bilayer. The steroid moiety was expected to act as the hydrophobic anchor for bilayer orientation and steric bulk was expected to prevent the polyamine penetrating the bilayer. Proton transport was assessed in unilamellar vesicles using the pH-fluorescence technique in which the external pH was increased to 7.8 relative to the internal pH at 7.4. The results demonstrated that 8 was almost as active as gramicidin, and maximal flux was achieved in ca. 20 s. 

Interaction with membrane sterols, formation of membrane channels... 

Dihydroceramide exhibits different physical and biochemical effects compared with the corresponding ceramide bearing the same A-acyl chain. For example, channel formation in mitochondrial membranes (78) and protein phosphatase activity in pancreatic beta cells are inhibited by dihydroceramide but are activated by ceramide (63, 79). Dihydroceramide is less effective than ceramide in inducing apoptosis (68). 

---