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Modulation of the conductance of a 2,2'-bipyridine-functionalized peptidic ion channel by Ni2+.

Pilz CS, Steinem C - Eur. Biophys. J. (2008)

Bottom Line: The first two conductance states appear much more prominent demonstrating that the complexation of bipyridine by Ni2+ results in a considerable confinement of the observed multiple conductance states.However, the conductance levels were independent of the presence of Ni2+.Moreover, from a detailed analysis of the open lifetimes of the channels, we conclude that the complexation of Ni2+ diminishes the frequency of channel events with larger open times.

View Article: PubMed Central - PubMed

Affiliation: Institut für Organische und Biomolekulare Chemie, Georg-August Universität, Tammannstr. 2, 37077, Göttingen, Germany.

ABSTRACT
An alpha-helical amphipathic peptide with the sequence H2N-(LSSLLSL)3-CONH2 was obtained by solid phase synthesis and a 2,2'-bipyridine was coupled to its N-terminus, which allows complexation of Ni2+. Complexation of the 2,2'-bipyridine residues was proven by UV/Vis spectroscopy. The peptide helices were inserted into lipid bilayers (nano black lipid membranes, nano-BLMs) that suspend the pores of porous alumina substrates with a pore diameter of 60 nm by applying a potential difference. From single channel recordings, we were able to distinguish four distinct conductance states, which we attribute to an increasing number of peptide helices participating in the conducting helix bundle. Addition of Ni2+ in micromolar concentrations altered the conductance behaviour of the formed ion channels in nano-BLMs considerably. The first two conductance states appear much more prominent demonstrating that the complexation of bipyridine by Ni2+ results in a considerable confinement of the observed multiple conductance states. However, the conductance levels were independent of the presence of Ni2+. Moreover, from a detailed analysis of the open lifetimes of the channels, we conclude that the complexation of Ni2+ diminishes the frequency of channel events with larger open times.

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Channel recordings of 3 in nano-BLMs. a Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the absence of NiCl2. b Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the presence of NiCl2. c Histogram analysis of the observed conductance states of 3 in nano-BLMs up to 600 pS [3,556 events, bin width: 5 pS, normalized to all events (4,524)] in the absence of NiCl2. d Histogram analysis of the conductance states of 3 in the presence of 2.5–5 μM NiCl2 up to a conductance of 600 pS [2,029 events, bin width: 5 pS, normalized to all events (2189)]. The solid lines are the results of fitting four Gaussian distributions (dotted lines) to the histograms
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Fig2: Channel recordings of 3 in nano-BLMs. a Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the absence of NiCl2. b Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the presence of NiCl2. c Histogram analysis of the observed conductance states of 3 in nano-BLMs up to 600 pS [3,556 events, bin width: 5 pS, normalized to all events (4,524)] in the absence of NiCl2. d Histogram analysis of the conductance states of 3 in the presence of 2.5–5 μM NiCl2 up to a conductance of 600 pS [2,029 events, bin width: 5 pS, normalized to all events (2189)]. The solid lines are the results of fitting four Gaussian distributions (dotted lines) to the histograms

Mentions: First, the ion channel properties of 3 in the absence of Ni2+ were characterized by single channel recordings under voltage clamp conditions. 3 was added from an ethanolic solution to the cis-compartment in a concentration of 0.03–3.75 nM, while applying a holding potential of +200 mV (referring to the cis side). After addition of the peptide, characteristic rectangular current traces were observed (Fig. 2a). We attribute this step-like current increase and decrease to the assembly and disassembly of a helix bundle composed of a defined number of amphipathic peptide helices. Altogether, four different opening levels were discernable in the current range of around 25 pA (O1), 35 pA (O2), 45 pA (O3) and 70 pA (O4). Channel events of one opening level were often observed in bursts, which lasted several seconds. The majority of all observed channel events were found in the range of 15–110 pA. To quantitatively evaluate the conductance levels, a conductance histogram analysis was performed. All events that were characterized by a current plateau were taken as channel events. Conductances were calculated from the current traces taking the applied voltage of +200 mV into account. Figure 2c shows the obtained histogram up to a conductance of 600 pS including 3,556 events. Four Gaussian functions were fitted to the data resulting in four distinct conductance states with G1 = (131 ± 19) pS, G2 = (181 ± 20) pS, G3 = (234 ± 20) pS, and G4 = (374 ± 70) pS. 968 events were found to exhibit a conductance state above 600 pS, which is 21% of all events that were read out (see supplementary material). Taking the fact that the lowest conductance state is a result of six peptide monomers forming a helix bundle, like it is proposed by DeGrado and coworkers (Akerfeldt et al. 1993; Lear et al. 1988), the higher conductance states would be a result of a continuous increase in the number of helices participating in the bundle up to nine helices. Similarly, we have found five different conductance states of alamethicin in nano-BLMs in 0.5 M KCl exhibiting conductance levels ranging from 200 to 6,000 pS (Römer and Steinem 2004). The different observed conductance states are attributed to helix bundles composed of different numbers of alamethicin monomers, ranging from 4 up to 11 (Sansom 1991).Fig. 2


Modulation of the conductance of a 2,2'-bipyridine-functionalized peptidic ion channel by Ni2+.

Pilz CS, Steinem C - Eur. Biophys. J. (2008)

Channel recordings of 3 in nano-BLMs. a Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the absence of NiCl2. b Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the presence of NiCl2. c Histogram analysis of the observed conductance states of 3 in nano-BLMs up to 600 pS [3,556 events, bin width: 5 pS, normalized to all events (4,524)] in the absence of NiCl2. d Histogram analysis of the conductance states of 3 in the presence of 2.5–5 μM NiCl2 up to a conductance of 600 pS [2,029 events, bin width: 5 pS, normalized to all events (2189)]. The solid lines are the results of fitting four Gaussian distributions (dotted lines) to the histograms
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Fig2: Channel recordings of 3 in nano-BLMs. a Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the absence of NiCl2. b Characteristic current trace (channel opens upwards) of 3 obtained at +200 mV in 0.5 M KCl in the presence of NiCl2. c Histogram analysis of the observed conductance states of 3 in nano-BLMs up to 600 pS [3,556 events, bin width: 5 pS, normalized to all events (4,524)] in the absence of NiCl2. d Histogram analysis of the conductance states of 3 in the presence of 2.5–5 μM NiCl2 up to a conductance of 600 pS [2,029 events, bin width: 5 pS, normalized to all events (2189)]. The solid lines are the results of fitting four Gaussian distributions (dotted lines) to the histograms
Mentions: First, the ion channel properties of 3 in the absence of Ni2+ were characterized by single channel recordings under voltage clamp conditions. 3 was added from an ethanolic solution to the cis-compartment in a concentration of 0.03–3.75 nM, while applying a holding potential of +200 mV (referring to the cis side). After addition of the peptide, characteristic rectangular current traces were observed (Fig. 2a). We attribute this step-like current increase and decrease to the assembly and disassembly of a helix bundle composed of a defined number of amphipathic peptide helices. Altogether, four different opening levels were discernable in the current range of around 25 pA (O1), 35 pA (O2), 45 pA (O3) and 70 pA (O4). Channel events of one opening level were often observed in bursts, which lasted several seconds. The majority of all observed channel events were found in the range of 15–110 pA. To quantitatively evaluate the conductance levels, a conductance histogram analysis was performed. All events that were characterized by a current plateau were taken as channel events. Conductances were calculated from the current traces taking the applied voltage of +200 mV into account. Figure 2c shows the obtained histogram up to a conductance of 600 pS including 3,556 events. Four Gaussian functions were fitted to the data resulting in four distinct conductance states with G1 = (131 ± 19) pS, G2 = (181 ± 20) pS, G3 = (234 ± 20) pS, and G4 = (374 ± 70) pS. 968 events were found to exhibit a conductance state above 600 pS, which is 21% of all events that were read out (see supplementary material). Taking the fact that the lowest conductance state is a result of six peptide monomers forming a helix bundle, like it is proposed by DeGrado and coworkers (Akerfeldt et al. 1993; Lear et al. 1988), the higher conductance states would be a result of a continuous increase in the number of helices participating in the bundle up to nine helices. Similarly, we have found five different conductance states of alamethicin in nano-BLMs in 0.5 M KCl exhibiting conductance levels ranging from 200 to 6,000 pS (Römer and Steinem 2004). The different observed conductance states are attributed to helix bundles composed of different numbers of alamethicin monomers, ranging from 4 up to 11 (Sansom 1991).Fig. 2

Bottom Line: The first two conductance states appear much more prominent demonstrating that the complexation of bipyridine by Ni2+ results in a considerable confinement of the observed multiple conductance states.However, the conductance levels were independent of the presence of Ni2+.Moreover, from a detailed analysis of the open lifetimes of the channels, we conclude that the complexation of Ni2+ diminishes the frequency of channel events with larger open times.

View Article: PubMed Central - PubMed

Affiliation: Institut für Organische und Biomolekulare Chemie, Georg-August Universität, Tammannstr. 2, 37077, Göttingen, Germany.

ABSTRACT
An alpha-helical amphipathic peptide with the sequence H2N-(LSSLLSL)3-CONH2 was obtained by solid phase synthesis and a 2,2'-bipyridine was coupled to its N-terminus, which allows complexation of Ni2+. Complexation of the 2,2'-bipyridine residues was proven by UV/Vis spectroscopy. The peptide helices were inserted into lipid bilayers (nano black lipid membranes, nano-BLMs) that suspend the pores of porous alumina substrates with a pore diameter of 60 nm by applying a potential difference. From single channel recordings, we were able to distinguish four distinct conductance states, which we attribute to an increasing number of peptide helices participating in the conducting helix bundle. Addition of Ni2+ in micromolar concentrations altered the conductance behaviour of the formed ion channels in nano-BLMs considerably. The first two conductance states appear much more prominent demonstrating that the complexation of bipyridine by Ni2+ results in a considerable confinement of the observed multiple conductance states. However, the conductance levels were independent of the presence of Ni2+. Moreover, from a detailed analysis of the open lifetimes of the channels, we conclude that the complexation of Ni2+ diminishes the frequency of channel events with larger open times.

Show MeSH
Related in: MedlinePlus