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Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform.

Saha SC, Henderson AJ, Powl AM, Wallace BA, de Planque MR, Morgan H - PLoS ONE (2015)

Bottom Line: The platform allows up to 6 bilayers to be analysed simultaneously.Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips.The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds.

View Article: PubMed Central - PubMed

Affiliation: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, United Kingdom.

ABSTRACT
This paper describes the use of a newly-developed micro-chip bilayer platform to examine the electrophysiological properties of the prokaryotic voltage-gated sodium channel pore (Na(v)Sp) from Silicibacter pomeroyi. The platform allows up to 6 bilayers to be analysed simultaneously. Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips. The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds. Dose-dependent modulation of the opening probability by the channel blocking drug nifedipine was measured and its IC50 determined.

No MeSH data available.


Related in: MedlinePlus

Ion channel events and their current voltage relationship.(a) Representative single channel current traces for the NavSp pore at different applied transmembrane potentials. (b) I-V plot for the NavSp pore. Error bars are one standard deviation for ≥ 3 individual bilayer measurements. The electrophysiology buffer is 0.5 M NaCl, 10 mM HEPES, pH 7.4. Channels were introduced in the aperture-suspended bilayer of POPE:POPG (1:1) by fusion of proteoliposomes with a lipid-to-protein ratio of 300:1. Data were recorded at a sampling frequency of 1.25 MHz and were low pass-filtered at 1.25 kHz.
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pone.0131286.g002: Ion channel events and their current voltage relationship.(a) Representative single channel current traces for the NavSp pore at different applied transmembrane potentials. (b) I-V plot for the NavSp pore. Error bars are one standard deviation for ≥ 3 individual bilayer measurements. The electrophysiology buffer is 0.5 M NaCl, 10 mM HEPES, pH 7.4. Channels were introduced in the aperture-suspended bilayer of POPE:POPG (1:1) by fusion of proteoliposomes with a lipid-to-protein ratio of 300:1. Data were recorded at a sampling frequency of 1.25 MHz and were low pass-filtered at 1.25 kHz.

Mentions: Bilayers were made using POPC and POPG lipids since previous ion flux studies for these pore-only constructs [10] showed that this lipid composition is suitable for observing active channels. Successful recordings were most frequently obtained from liposomes with 300:1 lipid-to-protein ratios. For a symmetrical salt concentration (0.5 M NaCl, 10 mM HEPES, pH 7.4), typical single channel events are shown in Fig 2(A). At this NaCl concentration, the single channel conductance was 106 pS. I-V plots are shown in Fig 2(B); no rectifying behaviour was observed. This is not unexpected since the pore-only construct does not contain the voltage sensor region of the channel. The electrical behaviour is generally similar to data from Shaya et al. [5] for the NavSp1 pore-only construct, inserted into giant unilamellar vesicles (GUVs) made of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) for planar patch clamp electrophysiology. The authors used asymmetric salt conditions (200 mM NaCl and 110 mM KCl) and measured channel conductances of ~40 pS. Full length NaChBac channels have also been characterized using both whole cell patch clamp [6] and bilayer lipid membranes [9]. The patch clamp method gave a channel conductance of 12 pS (8 mM and 130 mM NaCl), which is much less than determined from our data, but the lipid bilayer studies yielded conductance values of 120 pS for symmetric salt conditions (150 mM NaCl), very close to the values measured in this study for the pore. Our data indicates that NavSp is mostly open when inserted into the bilayer, but the opening time and open probability varies from experiment to experiment. This behaviour has also been observed by others, both for pore-only constructs [5] and for full length channels [6, 9].


Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform.

Saha SC, Henderson AJ, Powl AM, Wallace BA, de Planque MR, Morgan H - PLoS ONE (2015)

Ion channel events and their current voltage relationship.(a) Representative single channel current traces for the NavSp pore at different applied transmembrane potentials. (b) I-V plot for the NavSp pore. Error bars are one standard deviation for ≥ 3 individual bilayer measurements. The electrophysiology buffer is 0.5 M NaCl, 10 mM HEPES, pH 7.4. Channels were introduced in the aperture-suspended bilayer of POPE:POPG (1:1) by fusion of proteoliposomes with a lipid-to-protein ratio of 300:1. Data were recorded at a sampling frequency of 1.25 MHz and were low pass-filtered at 1.25 kHz.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493117&req=5

pone.0131286.g002: Ion channel events and their current voltage relationship.(a) Representative single channel current traces for the NavSp pore at different applied transmembrane potentials. (b) I-V plot for the NavSp pore. Error bars are one standard deviation for ≥ 3 individual bilayer measurements. The electrophysiology buffer is 0.5 M NaCl, 10 mM HEPES, pH 7.4. Channels were introduced in the aperture-suspended bilayer of POPE:POPG (1:1) by fusion of proteoliposomes with a lipid-to-protein ratio of 300:1. Data were recorded at a sampling frequency of 1.25 MHz and were low pass-filtered at 1.25 kHz.
Mentions: Bilayers were made using POPC and POPG lipids since previous ion flux studies for these pore-only constructs [10] showed that this lipid composition is suitable for observing active channels. Successful recordings were most frequently obtained from liposomes with 300:1 lipid-to-protein ratios. For a symmetrical salt concentration (0.5 M NaCl, 10 mM HEPES, pH 7.4), typical single channel events are shown in Fig 2(A). At this NaCl concentration, the single channel conductance was 106 pS. I-V plots are shown in Fig 2(B); no rectifying behaviour was observed. This is not unexpected since the pore-only construct does not contain the voltage sensor region of the channel. The electrical behaviour is generally similar to data from Shaya et al. [5] for the NavSp1 pore-only construct, inserted into giant unilamellar vesicles (GUVs) made of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) for planar patch clamp electrophysiology. The authors used asymmetric salt conditions (200 mM NaCl and 110 mM KCl) and measured channel conductances of ~40 pS. Full length NaChBac channels have also been characterized using both whole cell patch clamp [6] and bilayer lipid membranes [9]. The patch clamp method gave a channel conductance of 12 pS (8 mM and 130 mM NaCl), which is much less than determined from our data, but the lipid bilayer studies yielded conductance values of 120 pS for symmetric salt conditions (150 mM NaCl), very close to the values measured in this study for the pore. Our data indicates that NavSp is mostly open when inserted into the bilayer, but the opening time and open probability varies from experiment to experiment. This behaviour has also been observed by others, both for pore-only constructs [5] and for full length channels [6, 9].

Bottom Line: The platform allows up to 6 bilayers to be analysed simultaneously.Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips.The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds.

View Article: PubMed Central - PubMed

Affiliation: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, United Kingdom.

ABSTRACT
This paper describes the use of a newly-developed micro-chip bilayer platform to examine the electrophysiological properties of the prokaryotic voltage-gated sodium channel pore (Na(v)Sp) from Silicibacter pomeroyi. The platform allows up to 6 bilayers to be analysed simultaneously. Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips. The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds. Dose-dependent modulation of the opening probability by the channel blocking drug nifedipine was measured and its IC50 determined.

No MeSH data available.


Related in: MedlinePlus