Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform
July
Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform
Shimul Chandra Saha 0 1 2
Alexander J. Henderson 0 1 2
Andrew M. Powl 0 1 2
B. A. Wallace 0 1 2
Maurits R. R. de Planque 0 1 2
Hywel Morgan 0 1 2
0 1 Electronics and Computer Science, University of Southampton , Southampton, SO17 1BJ , United Kingdom , 2 Institute for Life Sciences, University of Southampton , Southampton, SO17 1BJ , United Kingdom , 3 Institute of Structural and Molecular Biology, Birkbeck College, University of London , London, WC1E 7HX , United Kingdom
1 Funding: This work was supported by a linked grant from the UK Engineering and Physical Sciences Research Council, EP/H044795/1 to HM and MRRDP and EP/H043888/1 to BAW
2 Editor: Zhe Zhang, Xuzhou Medical College , CHINA
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 (NavSp) 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.
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Competing Interests: The authors have declared
that no competing interests exist.
Human voltage-gated sodium channels (Nav's) are large pseudotetrameric integral membrane
proteins which play important roles in many physiological processes and are linked to
channelopathies including epilepsy, pain disorders and cardiac conditions such as long QT syndrome
and Brugada syndrome [1–3]. Consequently they are major targets for drug development, and
hence are of considerable interest to the pharmaceutical industry. Recombinant expression and
purification of human channels have proved difficult due to their size and complexity in terms
of post translational modifications. Prokaryotic voltage-gated sodium channels are simpler,
single domain tetrameric orthologues which are more easily overexpressed, purified and
reconstituted into lipid bilayers. Each monomeric subunit consists of 6 transmembrane helices
(S1-S6), with S1-S4 comprising the voltage-sensing region and S5-S6 the pore region. Here we
have expressed the isolated tetrameric pore-only construct of the prokaryotic voltage-gated
sodium channel NavSp from Silicibacter pomeroyi. This pore-only construct has previously
been shown to be correctly folded, more thermally stable than the full length channel and
capable of supporting sodium flux, and is inhibitable by known eukaryotic channel blockers [4,
5]. Previously patch clamp techniques have demonstrated ensemble averaged currents for
NaChBac, another voltage gated sodium channel orthologue, this time from Bacillus
halodurans. Its activity is capable of being modulated by the calcium channel blocker drugs nifedipine,
nimodipine and mibefradil [6]. Other orthologues such as the NavMs channel from
Magnetococcus marinus [7] and the NavAb channel from Arcobacter butzleri [8] have been
characterised by patch clamping of those channels expressed in HEK293 cells. Single channel
characterisations of the NavSp pore and NaChBac have been performed using bilayer lipid
membranes [5, 9], whilst sodium flux measurements of the NavSp pore and other channels and
pore-only constructs [10] have been done using reconstituted proteins in liposomes. However
to date, there is no data on the IC50 for any of these proteins at the single channel level when
reconstituted into artificial lipid bilayer membranes.
In this study, we use a miniature microfluidic ion-channel screening platform to
characterise the NavSp pore construct. Bilayers were made across cavities fabricated in a microfabricated
polymer support made on small glass chips. Each of these chips holds two independent bilayers
and a miniature platform has been developed to accommodate three chips, allowing six bilayers
to be continuously measured in parallel, as shown in Fig 1(A). The microfluidic chips interface
with custom-designed integrated amplifiers (ASICs) and recording hardware/software as
shown in Fig 1(B). The glass chips contain integrated Ag/AgCl electrodes together with edge
Fig 1. Bilayer platform for parallel channel recording. (a) Photograph of the BLM recording platform with three bilayer chips, each with two bilayers. (b) A
daughter board showing two dual core ASIC amplifiers interfacing with the microfluidic chips. The integrated platform comprises three identical daughter
boards, each of which can record from up to two bilayers, together with signal processing electronics. Data is transmitted to a computer via a USB
connection. (c) Close up view of the bilayer aperture with Ag/AgCl electrode and flow channel. (d) Cross section X-X’ of the ch (...truncated)