The pore-forming properties of native and synthetic alamethicins were investigated in photoreceptor rod outer segments (OS) isolated from frog retina, and recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS within less than 50 ms by means of a computer-controlled micro-perfusion system. Once blocked with light, the main OS endogenous conductance, the OS membrane resistance was >1 GOmega, allowing low-noise and high-resolution recordings. Currents of ca. 700 pA were recorded in symmetric K(+) (100 mM) and Ca(2+) (1 mM), upon applying 1 microM of alamethicin F50/5 or its [L-Glu(OMe)(7,18,19)] analogue to the OS membrane (clamped at -20 mV). In the latter peptide, the Gln residues at positions 7, 18, and 19 were substituted with side-chain esterified Glu residues. For both peptides, the current activated exponentially, with a delay from peptide application, and exponentially returned to zero without any delay, upon removing the peptide from the external solution. The delay as well as the activation (tau(a)) and deactivation (tau(d)) time constants of the current produced by the modified alamethicin were much slower, and the current noise was much larger, with respect to the corresponding values for alamethicin F50/5. Therefore, the above three Gln residues are not a key factor for pore formation, but the [L-Glu(OMe)(7,18,19)] analogue produces larger pores with a lower probability of formation.

Pore-forming properties of alamethicin F50/5 inserted in a biological membrane

VEDOVATO, Natascia;RISPOLI, Giorgio
2007

Abstract

The pore-forming properties of native and synthetic alamethicins were investigated in photoreceptor rod outer segments (OS) isolated from frog retina, and recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS within less than 50 ms by means of a computer-controlled micro-perfusion system. Once blocked with light, the main OS endogenous conductance, the OS membrane resistance was >1 GOmega, allowing low-noise and high-resolution recordings. Currents of ca. 700 pA were recorded in symmetric K(+) (100 mM) and Ca(2+) (1 mM), upon applying 1 microM of alamethicin F50/5 or its [L-Glu(OMe)(7,18,19)] analogue to the OS membrane (clamped at -20 mV). In the latter peptide, the Gln residues at positions 7, 18, and 19 were substituted with side-chain esterified Glu residues. For both peptides, the current activated exponentially, with a delay from peptide application, and exponentially returned to zero without any delay, upon removing the peptide from the external solution. The delay as well as the activation (tau(a)) and deactivation (tau(d)) time constants of the current produced by the modified alamethicin were much slower, and the current noise was much larger, with respect to the corresponding values for alamethicin F50/5. Therefore, the above three Gln residues are not a key factor for pore formation, but the [L-Glu(OMe)(7,18,19)] analogue produces larger pores with a lower probability of formation.
Vedovato, Natascia; Baldini, C; Toniolo, C; Rispoli, Giorgio
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/520689
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