A novel technique to study pore-forming peptides in a natural membrane.
The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na(+):Ca(2+),K(+) exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K(+), Na(+) or Ca(2+)), the OS membrane resistance (R ( m )) was typically larger than 1 GOmega in the presence of 1 mM external Ca(2+). Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in approximately 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 were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.
Vedovato
Natascia
N
CNISM, Dipartimento di Biologia ed Evoluzione, Sezione di Fisiologia e Biofisica and Centro di Neuroscienze, Università di Ferrara, via Borsari 46, Ferrara, Italy.
Rispoli
Giorgio
G
D000818:Animals; D002118:Calcium / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D008027:Light; D010455:Peptides / Q000737:chemistry* / Q000502:physiology; D011188:Potassium / Q000378:metabolism; D011893:Rana esculenta / Q000502:physiology*; D012374:Rod Cell Outer Segment / Q000502:physiology*; D012964:Sodium / Q000378:metabolism
D007473:Ion Channels; D010455:Peptides; D012964:Sodium; D006152:Cyclic GMP; D011188:Potassium; D002118:Calcium
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