Guidelines for the development of direct vesicular transport assays

The successful detection of the transport of a compound in the direct vesicular transport assay is dependent on a number of properties of the compound assayed:

Permeability: In order to measure ATP dependent accumulation of the substrate candidate one should assume that the compound has low enough permeability to avoid its passive diffusion out of the vesicles. Highly and moderately permeable compounds (P_app PAMPA > 10⁶ cm/s) will diffuse out of the vesicles thereby making the transporter function undetectable.
Sensitive analytics: The volume of the vesicles is very low, therefore, the amount of trapped compound is very low (in the pmols range). There should be an existing cold analytical method (HPLC, LC-MS) for the sensitive detection of the substrate candidate. The preparation of a calibration curve using this analytical method is advisable to determine the lowest amount of sample that can be unambiguously detected.
Affinity of the compound to the transporter: Active transport processes are saturated at high concentrations, while passive processes (e.g. binding of the compound to the vesicle, filter, etc.) are not. Therefore, choosing saturating concentrations of the compound tested might result in high background which makes the active transport undetectable. Studies should be performed at around the affinity of the substrate candidate for the transporter of interest.

Based on these we suggest using the following general guideline for conducting such study:

Determine the passive permeability of your compound by any adequate method (e.g. HDM-PAMPA).
Establish a sensitive analytical method that is able to measure picomoles of the compound. Radiolabeled compounds are very helpful in these transport studies. Alternatively, a sensitive HPLC/MS or LC/MS/MS method should be set up.
In an alternative assay (can be indirect vesicular transport study or any other suitable transporter assay) confirm that there is an interaction between the substrate candidate and the transporter of interest and determine the affinity (e.g. IC₅₀).
Around the IC₅₀ value obtained choose two concentrations of the substrate candidate (one lower and one higher than the IC₅₀) and measure the ATP-dependent accumulation with time for a shorter (e.g. two minutes) and a longer period (e.g. 20 minutes). Repeat this experiment on control membrane vesicles recommended by SOLVO. If reproducible, this type of assay should give you a yes-or-no type answer whether your compound is a transported substrate of the given transporter.
In case K_m and V_max value determination is necessary, the transport experiment should be repeated with increasing concentrations of the substrate candidate in the presence and absence of ATP for a time period chosen in the previous experiment.

Matter of transporter specificity. Transporters of the ABCC family (MRPs) usually transport anionic compounds that are usually of modest permeability, making them good substrates in the vesicular transport assay. More hydrophobic MRP substrates may be cotransported with GSH, so the addition of GSH to the systems should be considered. ABCG2 (BCRP/MXR) transports both hydrophobic and hydrophilic compounds. In case of investigating substrate candidates for this efflux transporter one should consider this. A PAMPA assay to evaluate the passive permeability of the compound is necessary. The physiological function of ABCB11 (BSEP) is bile salt transport. Little is known whether BSEP transports known cholestatic compounds or whether these act only as an inhibitor of BSEP function. ABCB1 (MDR1) mainly transports hydrophobic compounds and some cationic ones. The investigator would run into the most problems trying to identify MDR1 substrates, using the vesicular transport assay. We recommend alternative assays, preferably monolayer assays for the determination of the substrate nature of such compounds.

Figures

Figure 1. Difference between transport values measured in the presence and in the absence of ATP. A reproducible, more than 2-fold difference is an indication that the test compound is a transported substrate.

Figure 2. Time dependence of substrate accumulation in the vesicles. For further experiments a time point from the linear phase (indicated by a blue dotted line) of the curve should be chosen (in this case the 4-minute point was chosen).

Figure 3. Concentration dependence of substrate transport. Many cases the transport can be modeled using the Michaelis-Menten equation (A). Some substrates are transported via a more complex mechanism involving 2 or more binding sites. In this case a sigmoid type curve is observed (B).