The biophysical characteristics and the pore formation dynamics of synthetic, naturally occurring peptides forming membrane-spanning channels were investigated by using isolated rod outer segments (OS) of reptilia and amphibia recorded in whole-cell configuration. The peptides were applied (and removed) to the OS in <50 ms with a computer-controlled microperfusion system. Once blocking the two OS endogenous conductances (the cGMP channels by light and the retinal exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K+, Na+ or Ca2+), the OS membrane resistance (Rm) could be >5 GΏ. Therefore, any exogenous current can be studied down to the single channel level. Macroscopic currents of amplitude of ~300 pA were recorded in symmetric K+ or Na+ (>100 mM) and Ca2+ (1 mM) from the synthetic, naturally occurring alamethicin F50/5 and selected analogs applied at 1 μM concentration at –20 mV. Once applying and removing the peptide, the current activated and deactivated with a time constant of about 160 ms. The synthetic analogs Glu(OMe)7,18,19 and Glu(OMe)18,19 of the F50/5 produced a current of about 100 pA at 1 μM concentration, and they showed a strong activation by hyperpolarization as alamethicin F50/5. Clear single channel events were observed when the concentration of all the above peptides was reduced to <250 nM. However, no current was recorded from the synthetic nOtt-(11)-OMe trichogin at 1 μM for voltages up to +/- 60 mV. These results indicate that the Gln residues at positions 7, 18 and 19 of alamethicin are not a key factor for pore formation and its conduction properties. In general, the pore assembly and disassembly are very fast and cooperative events.

Biophysical Properties of Alamethicin F50/5 and Selected Analogues Inserted in Rod Outer Segment Membranes

VEDOVATO, Natascia;RISPOLI, Giorgio
2007

Abstract

The biophysical characteristics and the pore formation dynamics of synthetic, naturally occurring peptides forming membrane-spanning channels were investigated by using isolated rod outer segments (OS) of reptilia and amphibia recorded in whole-cell configuration. The peptides were applied (and removed) to the OS in <50 ms with a computer-controlled microperfusion system. Once blocking the two OS endogenous conductances (the cGMP channels by light and the retinal exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K+, Na+ or Ca2+), the OS membrane resistance (Rm) could be >5 GΏ. Therefore, any exogenous current can be studied down to the single channel level. Macroscopic currents of amplitude of ~300 pA were recorded in symmetric K+ or Na+ (>100 mM) and Ca2+ (1 mM) from the synthetic, naturally occurring alamethicin F50/5 and selected analogs applied at 1 μM concentration at –20 mV. Once applying and removing the peptide, the current activated and deactivated with a time constant of about 160 ms. The synthetic analogs Glu(OMe)7,18,19 and Glu(OMe)18,19 of the F50/5 produced a current of about 100 pA at 1 μM concentration, and they showed a strong activation by hyperpolarization as alamethicin F50/5. Clear single channel events were observed when the concentration of all the above peptides was reduced to <250 nM. However, no current was recorded from the synthetic nOtt-(11)-OMe trichogin at 1 μM for voltages up to +/- 60 mV. These results indicate that the Gln residues at positions 7, 18 and 19 of alamethicin are not a key factor for pore formation and its conduction properties. In general, the pore assembly and disassembly are very fast and cooperative events.
Channel forming peptides; Patch clamp; Peptaibols; Rod photoreceptor
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1378619
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