The pore forming properties of native and synthetic alamethicins were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS in <50 ms with a computer-controlled microperfusion system. Once blocked with light the main OS endogenous conductance, the OS membrane resistance was >1 GΩ, allowing low-noise and high resolution recordings. Currents of ~700 pA were recorded in symmetric K+ (100 mM) and Ca2+ (1 mM), upon applying to the OS membrane (clamped at –20 mV) 1 μM concentration of alamethicin F50/5 and its [L-Glu(OMe)7,18,19] analog. The latter had the Gln residues at positions 7, 18 and 19 substituted with Glu, characterized by a methyl ester group linked to the carboxyl function in the γ position. 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 and the activation and deactivation time constants of the current produced by the [L-Glu(OMe)7,18,19] analog were much slower, and the current noise was much larger, with respect to those of 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] analog 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
2009
Abstract
The pore forming properties of native and synthetic alamethicins were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS in <50 ms with a computer-controlled microperfusion system. Once blocked with light the main OS endogenous conductance, the OS membrane resistance was >1 GΩ, allowing low-noise and high resolution recordings. Currents of ~700 pA were recorded in symmetric K+ (100 mM) and Ca2+ (1 mM), upon applying to the OS membrane (clamped at –20 mV) 1 μM concentration of alamethicin F50/5 and its [L-Glu(OMe)7,18,19] analog. The latter had the Gln residues at positions 7, 18 and 19 substituted with Glu, characterized by a methyl ester group linked to the carboxyl function in the γ position. 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 and the activation and deactivation time constants of the current produced by the [L-Glu(OMe)7,18,19] analog were much slower, and the current noise was much larger, with respect to those of 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] analog produces larger pores with a lower probability of formation.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
