Enhanced Patch-Clamp Technique to Study Antimicrobial Peptides and Viroporins, Inserted in a Cell Plasma Membrane with Fully Inactivated Endogenous Conductances

Many short peptides selectively permeabilize the bacteria plasma membrane, leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. Another class of short (<100 residues) membrane proteins called viroporins, because they are coded by viral genes, permeabilizes the membrane of susceptible cells during infection of by most animal viruses. The permeabilization leads to host cell lyses and the release of the virus mass, replicated at host cell expense, to propagate the infection. Detailed knowledge of the permeabilization properties of these proteins would allow to design, for instance, selective blockers of these pores, that would contrast the spread of the viral infection. In this chapter, the patch-clamp technique is employed to study the mechanism of membrane permeabilization induced by the pore-forming peptides, under strict physiological conditions. This goal is achieved by recording the ion current through the channels formed by these peptides, once inserted in a cell plasma membrane. To avoid contamination by the cell membrane currents, all the endogenous current sources must be blocked. It has been found that the photoreceptor rod outer segment mechanically isolated from the retina of low vertebrates (OS) was the most suitable cell to carry on the above studies, because it was possible to fully block all its endogenous currents without using any drug (such as TTX, TEA, dihydropyridines, etc.), that could obstruct the peptide pores or interfere with the pore formation. The peptides were applied to (and removed from) the extracellular OS side in ~50 ms with a computer-controlled microperfusion system, in which every perfusion parameter (as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles) was controlled by a user-friendly interface. This system allowed rapid application and removal of ions, drugs and peptides on the cells with a controlled timing, so that the ion channel characteristics (as its selectivity, blockade and gating) and the dynamics of pore formation could be precisely assessed. On the basis of the electrophysiological recordings obtained with representative peptides and with selected analogs, as alamethicin F50/5, the cecoprine-mellitin hybrid peptide, and a 20-aminoacid long fragment of the viroporin poliovirus 2B, it will be shown that the membrane pore formation occurs according to the barrel and stave, toroidal, and carpet model, respectively, that are the most widely-accepted mechanisms of membrane permeabilization. When recording large currents (produced for instance by high concentrations of peptides and/or highly permeable peptides), it is necessary to minimize series resistance, to reduce time constant of charging the cell membrane capacitance and error in membrane potential control. A second problem arises from the asymmetry of the plasma membrane: it is possible that the permeabilization properties of a particular peptide could be different depending upon the side of the membrane to which it is applied. For example, it is conceivable that viroporins are optimized to insert in the intracellular face of the plasma membrane, because they are synthesized in host cell cytosol. These two problems could be circumvented by widening the patch pipette shank, through the calibrated combination of heat and air pressure. These pipettes dramatically reduce series resistance, and allow at the same time to insert pulled quartz or plastic tubes very close to the pipette tip, making it possible the delivery of large molecules to the cytosol with a controlled timing. Finally, it is presented here a simple procedure to consistently attain seals with conventional or pressure polished pipettes, made from just one glass type, on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes made with many different lipid mixtures.