Membrane transporter assay and methods of use
OwnerJacob Kellerinventor
Life of this patent
Estimating the term…
Abstract
This document provides materials and methods for a membrane transporter assay (e.g., an oscillating stimulus transporter assay (OSTA)). OSTA can be used to obtain temporal readouts of transporter activity and to screen for membrane transporter modulators (e.g., agonists or antagonists).
Claims42
A method of characterizing membrane transporter function, said method comprising:
- a) expressing a sensor in a cell comprising a membrane transporter, wherein the sensor capable of detecting a substrate transported by the membrane transporter;
- b) perfusing the cell with a solution comprising the substrate, wherein said solution changes or oscillates between a low substrate concentration and a high substrate concentration;
- and
- c) detecting the substrate, wherein changes in detection of the substrate can be used to characterize membrane transporter function.
A method of characterizing Cl−/HCO3− exchange across a cell membrane, said method comprising:
- a) expressing a fluorescent H+ sensor in a cell comprising a Cl−/HCO3− exchange transporter, wherein the fluorescent H+ sensor comprises a SNARF-5F-AM dye;
- b) perfusing the cell with a solution comprising Cl−, wherein said solution oscillates between about 8 mM Cl− and about 158 mM Cl−;
- and
- c) detecting the H+, wherein changes in the H+ can be used to characterize Cl−/HCO3− exchange across a cell membrane.
A method of characterizing SO42−/OH− exchange, said method comprising:
- a) expressing a fluorescent H+ sensor in a cell comprising a SO42+/OH− exchange transporter, wherein the fluorescent H+ sensor comprises a SNARF-5F-AM dye;
- b) perfusing the cell with a solution comprising SO42−, wherein said solution oscillates between about 0 mM SO42− and about 100 mM SO42−;
- and
- c) detecting H−, wherein changes in the H+ can be used to characterize SO42'1/OH− exchange.
A method of characterizing glucose transporter function, said method comprising:
- a) expressing a fluorescent glucose sensor in a cell comprising a glucose transporter, wherein the fluorescent glucose sensor is a cytosolic ratiometric peptide;
- b) perfusing the cell with a solution comprising glucose, wherein said solution oscillates between about 0 mM glucose and about 2 mM glucose;
- and
- c) detecting the glucose, wherein changes in detection of the glucose can be used to characterize glucose transporter function.
A method of characterizing glutamate transporter function, said method comprising:
- a) expressing a fluorescent glutamate sensor in a cell comprising a glutamate transporter, wherein the fluorescent glutamate sensor comprises iGluSnFR;
- b) perfusing the cell with a solution comprising glutamate, wherein said solution oscillates between about 0 mM glutamate and about 10 mM glutamate;
- and
- c) detecting the glutamate, wherein changes in detection of the glutamate can be used to characterize glutamate transporter function.
A method of identifying a substrate of a membrane transporter, said method comprising:
- a) expressing a sensor in a cell comprising a membrane transporter, wherein the sensor can detect a candidate substrate;
- b) perfusing the cell with a solution comprising the candidate substrate, wherein said solution oscillates between a high candidate substrate concentration and a low candidate substrate concentration;
- and
- c) detecting transport of the candidate substrate, wherein transport of the candidate substrate identifies the candidate substrate as the substrate of the membrane transporter.
A method of screening for modulators of membrane transporter function, said method comprising:
- a) expressing a sensor in a cell comprising a membrane transporter, wherein the sensor can detect a substrate transported by the membrane transporter;
- b) perfusing the cell with a solution comprising the substrate, wherein said solution oscillates between a low substrate concentration and a high substrate concentration;
- c) providing the cell with a candidate modulator;
- and
- d) detecting transport of the substrate, wherein a change in transport of the substrate identifies the candidate modulator as a modulator of membrane transporter function.
Dependencies are inferred from the claim text.