We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.
Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for “off-target” drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential “off-target”, effects. We find that results using this method are in good agreement with previous electrophysiological results 12.
1. Generate ANTS-filled Liposomes
2. Mix Fluorescence Solution
3. Setting up the Fluorescence Instrument
4. Doing an Experiment
5. Analyzing Data
6. Representative Results
Figure 1: The essentials of the fluorescence quench-based assay to detect changes in lipid bilayer properties. Top left: A zoom-in on a single lipid vesicle with ANTS plus NaNO3 on the inside and NaNO3 plus TlNO3 on the outside. Top right: The fluorescence signal recorded using (form top to bottom) ANTS-filled vesicles without quencher, with quencher, with quencher and pre-doped with 87, 260 and 780 nM gramicidin. Bottom: Schematic representation of the stopped-flow mixing chamber.
Figure 2: A screen shot from the Pro-Data SX software illustrating the various panels referenced in the description of the experimental setup.
Figure 3: Multiple repeat determinations of the fluorescence signal from ANTS-loaded LUVs with Na-buffer. The first four repeats are always excluded, as they contain mixing artifacts. The tubing connecting the sample syringes to the mixing cell have a defined volume, therefore the first few repeats will give us a reading of what was previously in the tubing: water for repeat 1 and 2, some combination of water and sample for repeat 3, mostly sample for repeat 4, and just sample for the remaining repeats.
Figure 4: Multiple repeat determinations of the fluorescence signal from ANTS-loaded LUVs with Na-buffer and with Tl-quencher. The first four repeats have been removed from both conditions. Additionally, for the Tl-quencher measurements, repeat 8 needs to be removed due to artifacts, most likely air bubbles.
Figure 5: Effect of capsaicin (Cap) on the time course of ANTS fluorescence quenching. (A) Normalized fluorescence signal over 1 s, gray dots denote results from all repeats (n > 5 per condition); red lines denote the average of all repeats. (B) The first 100 ms, gray dots denote results from a single repeat for each condition; red lines are stretched exponential fits (2 – 100 ms) to those repeats. The stippled blue line denotes the 2 ms mark, the time at which the rate of quenching is determined. In both A and B the top trace shows results in the absence of Tl+; the next two traces show results in the absence of gA, with Tl+ ± Cap; the four lower traces show results with 260 nM gA and Tl+, where the numbers denote [Cap] in μM. The rates of quenching for 0, 10, 30 and 90 μM Cap, as determined by the rate of a stretched exponential, are 36±6, 69±6, 85±8 and 247±27 (mean ± s.d., n > 8), respectively.
We have demonstrated a fast fluorescence-based assay for determining the bilayer modifying potential of drugs and other small amphiphiles. Compounds that modify bilayer properties are likely to alter membrane protein function in an indirect, nonspecific manner, possibly contributing to “off-target” drug effects. The assay exploits the power of gramicidin channels as probes for changes in bilayer properties 12 that are sensed by bilayer-spanning proteins. The results obtained using the fluorescence-based assay are in good agreement with results from single-channel gA experiments 12, indicating that this method can be used for mechanistic studies as well as for screening compound libraries. Using the present configuration of the assay we can test dozens of compounds a day, which is one-to-two orders of magnitude higher throughput than possible using the single-channel approach. There are no fundamental rate-limiting steps in the fluorescence-based assay, meaning that it can be extended to run in true high-throughput mode. It is possible to vary the assay’s electrolyte solutions and/or use different lipid composition for the LUVs’. It is worth noting that some lipid compositions can separate when dried, requiring rapid solvent exchange systems instead of drying/hydration steps 7.
It remains unclear whether there is a causal relationship between increasing lipophilicity of drug leads and increasing attrition in drug development 10,11,17 and, if so, what are the underlying mechanism(s)? Nevertheless, because membrane proteins tend to be regulated by changes in their membrane environment 2, it would be prudent to test whether amphiphilic drugs and drug leads alter pertinent lipid bilayer properties and, if so, at what concentrations? Amphiphile adsorption to the lipid bilayer will deplete the aqueous phase, therefore the relevant concentration is the free concentration in the aqueous phase which may be orders of magnitude less than the nominal concentration in the system, e.g. 6,9,16. If a molecule’s desired (biological) effects occur at concentrations where it alters bilayer properties, it becomes important to distinguish between the “non-specific”, bilayer-mediated changes in membrane protein function, as opposed to direct effects due to (high-affinity) binding to one or more target proteins. Knowing the bilayer-modifying propensity of a drug lead, discovered via conventional high-throughput screening, is therefore likely to be important for decisions regarding its further development.
Any amphiphile will at some concentration alter some bilayer property. Key considerations therefore become: at what concentration; and are the changes in bilayer properties sensed by (bilayer-spanning) membrane proteins? Here we exploit the ability of gramicidin to form channels by transbilayer dimerization 15. This makes them useful probes for the energetic coupling between lipid bilayers and bilayer-embedded proteins, and for exploring whether small molecules alter bilayer properties that are sensed by membrane proteins. The assay is fast, reliable and scalable, and therefore suitable for both biophysical studies and for screening compound libraries for drugs with potential bilayer-perturbing effects.
For additional information on the assay, verification of the assay’s effectiveness, and comparison with the single-channel electrophysiology see 12.
The authors have nothing to disclose.
We thank Michael J. Bruno, Radda Rusinova and Jon T. Sack for many stimulating discussions. Financial support from NIH, R01GM021342 and ARRA supplement R01GM021342-35S1, and the Josiah Macy, Jr. Foundation to OSA; the Tri-I CMB program for HII; and The Iris L. and Leverett S. Woodworth Medical Scientist Fellowship and NIH MSTP grant GM07739 for RK.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
ANTS | Invitrogen | A-350 | ||
gramicidin | Sigma Chemical Co | G-5002 | ||
1,2-dierucoyl-sn-glycero-3-phosphocholine | Avanti Polar Lipids | 850398C | ||
Mini-Extruder kit | Avanti Polar Lipids | 610000 | ||
PD-10 Desalting column | Sigma-Aldrich Made by GE Healthcare | 54805 |