Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Summary

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Abstract

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.

The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.

High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.

The chip technology is currently based on SiO₂ surfaces, but further functionalization using gold-coated chip surfaces is also possible.

Introduction

The analysis of membrane proteins has become of increasing interest for basic and pharmaceutical research in the past 20 years. The development of novel drugs depends on the identification and detailed characterization of new targets, currently being one of the limiting factors. The fact that about 60% of all drug targets are membrane proteins¹, makes the development of techniques to elucidate their function most important.

In the past, techniques for the study of electrogenic channels and transporters have been developed in multitude^2-4. Non-electrogenic substrates in contrary present a more chal....

Protocol

1. Preparation of Large Unilamellar Vesicles (LUVs)

1. Clean a round bottom flask (10 ml volume) with nitrogen gas to remove any dust particles. Rinse the round bottom flask with ethanol.
   Note: Residual ethanol can be tolerated and does not disturb subsequent steps.
2. Wash a 1 ml glass syringe 4x with chloroform, to remove any contamination. Add 4 ml SoyPC20 (25 mg/ml in chloroform) to the round bottom flask and dope the lipid solution with an additional DOPE^ATTO390 to a final concentration of 0.1 mol%.
   Note: Instead of DOPE^ATTO390 other fluorophore-labeled lipids or lipophilic dyes can also be....

Results

Pore-spanning membranes can easily be created on the nanostructured chip surface in a self-assembled manner. However the underlying process is still delicate and influenced by many parameters like liposome size, monodispersity of the liposome population, lamellarity, lipid- and salt-concentration and chemical surface properties. Most of these parameters have been carefully characterized and standardized in the above protocol. Other parameters however should be checked during every new pre.......

Discussion

The technique presented here allows a highly parallel analysis of membrane protein transport. Reconstituted membrane protein systems can directly be applied to the biochip, making the adaption of theoretically every membrane transporter or channel possible. Transport analysis is only limited by the establishment of a fluorescent read-out system, either via direct fluorescence change (translocation of fluorophores or fluorescently labeled substrates) or indirect fluorescence change (pH-sensitive dyes, secondary enzymatic .......

Materials

Name	Company	Catalog Number	Comments
Reagent
[2-(Trimethylammonium)ethyl] methanethiosulfonate	Toronto Research Chemicals Inc.	T792900	MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 °C. Use immediately (30 minutes) after solubilization in buffer.
1 ml gas-tight syringe	Hamilton	#1001
10 ml round flask	Schott Duran
2.7 mm glas beads	Roth	N032.1
2-Propanole	Roth	9866.5
30 cm Luer-Lock Extension Tube	Sarstedt	744304
Acetone	Roth	5025.5
Bio-Beads SM-2 Adsorbent	Bio-Rad	152-3920	need to be activated before first use
CaCl2 Dihydrat	Roth	HN04.3
Calcein	Sigma	C0875	store dark at -20 °C
Chloroform reagent grade	VWR Chemicals	22711324
DOPEATTO390	ATTO-TEC	AD 390-165	store dark at -20 °C
Ethanol absolute	Sigma-Aldrich	32205
Injekt Single-use syringe	Braun	460 60 51V
Injekt-F single-use syringe	Braun	91 66 017V
Keck clips	Schott	KC29
L-α-Phosphatidylcholine, 20% (Soy)	Avanti Polar Lipids	5416016	store under inert gas at -20 °C
NaCl 99.5% p.a.	Roth	3957.2
Nanopore E100 wafer/chips	Micromotive (Mainz/Germany)		available on request
Nucleopore Track-Etch Membrane 0.4 µm	Whatman	800282
Oregon Green Dextran 488 (70 kDa)	life Technologies	D-7173	store dark at -20 °C
Oy647	Luminartis (Münster/Germany)	OY-647-T-1mg	store dark at -20 °C
Rotilabo-syringe filtration, unsterile, pore-size 0.22 µm	Roth	P 818.1
Sephadex G-50	Sigma-Aldrich	G5080	column material for size exclusion chromatography
Silastic MDX4-4210	Dow Corning		curing agent for chip fixation onto cover glass support
sticky-Slide 8-well	ibidi	80828	multi-well chamber for the mounting onto glass slides (chip holder)
Three-way stopcock blue	Sarstedt	744410001
Tris Pufferan 99.9% Ultra Quality	Roth	5429.2
Triton-X 100	Roth	6683.1
Whatman 0.2 µm cellulose nitrate membrane filter	Roth	NH69.1
Name	Company	Catalog Number	Comments
Equipment
Büchi 461 water bath	Büchi
Büchi Rotavapor RE 111	Büchi
Cary Eclipse Fluorescence Spectrophtometer	Varian
LiposoFast Mini Extruder	Avestin
Membrane pump	Vaccubrand	15430
Nanosight Nanoparticle Tracking Microscope	Malvern / Nanosight	LM 14C
NyONE microscope	Synentec		available on request
Pump control	Vaccubrand	CVC 2II
Sonicator bath Sonorex RK100H	Brandelin electronic	31200001107477
Vaccum pump RC5	Vaccubrand	1805400204
Water bath W13	Haake	002-9910
Plasma Cleaner PDC-37G	Harrick Plasma	PDC-37G
Name	Company	Catalog Number	Comments
Software
ImageJ	Open Source	http://imagej.nih.gov/ij/	scientific image processing software
NanoCalcFX	Freeware	http://sourceforge.net/projects/nanocalc/	data analysis/evaluation software for massive transport kinetic datasets
NTA 2.3 Analytical Software	Nanosight		data acquisition and analysis software for nanoparticle tracking microscope
NTA 2.3 Temperature Comms	Nanosight		temperature controle software for nanoparticle tracking microscope