Permeabilization Alpha-toxin

Secretion. Exocytotic liberation of granular constituents has been demonstrated in leukocytes, platelets and neurological cells. Platelets attacked by alpha-toxin secrete platelet factor 4 and factor 5 (Arvand et a/., 1990 Bhakdi ef al., 1988). Release of the latter leads to assembly of platelet-bound prothrombinase complexes that generate thrombin. Alpha-toxin can thus promote coagulation via its perme-abilizing action on platelets. Release of granule constituents from leukocytes has been observed after permeabilization of these cells by HlyA. For example, large amounts of elastase are secreted into the extracellular medium (Bhakdi eta/., 1989). 

When very small pores that are permissive for monovalent but not for divalent ions are required, alpha-toxin should be used in a concentration range of 0.5-5 ng/ml. Most cells will become perme-abilized.Note, however, that certain cells exhibit a natural resistance towards alpha-toxin and pore formation will not occur. A simple means to discern whether permeabilization has taken place is to observe whether the cells swell increases in cell volume are the consequence of an uncontrolled flux of monovalent ions and water, and can be observed microscopically or by flow cytometry. Another simple method is measurement of cellular ATP. One hour after toxin application, cells are lysed with Triton X-100, and ATP is quantified using the luciferase assay (method described in (Bhakdi ef al., 1989)). ATP depletion will always be found in cells that have been permeabilized. 

Permeabilized cells allow the study of intracellular processes in situ under conditions which are believed to be close to the physiological situation in intact cells. Permeabilization by bacterial pore-forming toxins, alpha-toxin and streptolysin O (SLO) is now a widely accepted approach in the functional analysis of intracellular organelles. 

Alpha-toxin permeabilizes cells for small molecules (<3kDa) whereas SLO renders cells permeable for both small and large molecules (> 150 kDa). In this respect SLO resembles digitonin (Ahnert-Hilger eta/., 1989a,b 1993 Bhakdi efal., 1993). 

The experimental procedures for both PC 12 and adrenal chromaffin cells, using alpha-toxin or SLO to permeabilize the plasma membrane, are given below. 

The secretory response decreases with time after permeabilization. The amount of the decrease differs between different cells and preparations. In SLO-permeabilized PC 12 cells, exocytosis can be restored using cytosolic fractions. The free Ca concentration necessary to elicit exocytosis varies between 1 and 10 [xM depending on the cell preparations, but is always 3-5 fold higher in alpha-toxin-permeabilized cells. Since the permeability to Ca is the same in cells treated with alpha-toxin, SLO or digitonin (Fohr etal., 1989, 1991) buffering by intracellular proteins may be the reason tor this variability. 

PC 12 cells were permeabilized with purified SLO as given in section 18.3.1 of this chapter. Protein content was measured using the BCA-method. Values are the mean of three samples SD. Similar results were obtained using either wild-type SLO fusion protein or its alanine mutant. The assay can also be performed using alpha-toxin for permeabilization. 

Lind I, Ahnert-Hilger G, Fuchs G et at. (1987) Purification of alpha-toxin from staphylococcus aureus and application to cell permeabilization. Anolyt. Biochem. 164 84-89. 

Kawamoto S, Adelstein RS (1987) Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study. J Biol Chem 262 7282-7288 Kawase T, van Breemen C (1992) Aluminium fluoride induces a reversible Ca -sensi-tization in alpha-toxin permeabilized vascular smooth muscle. Eur J Pharmacol 214 39-44... 

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