Monitoring and manipulating cellular transmembrane potentials using nanostructures

Abstract

The use of nanostructures to monitor or modulate changes in cellular membrane potentials is disclosed. Nanoparticles having phospholipid coatings were found to display improved responses relative to nanoparticles having other coatings that do not promote localization or attraction to membranes.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application claims priority to U.S. Provisional Patent Application Ser. No. 60 / 659,975 filed Mar. 8, 2005, the contents of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention relates to compositions and methods useful for monitoring and manipulating cellular transmembrane voltages. In particular, nanoparticles and their use in monitoring and manipulating transmembrane voltages is disclosed.DESCRIPTION OF RELATED ART[0003]All cells have phospholipid membranes that serve as bimolecular barriers and to separate cell contents from the extracellular environment. The purpose of the plasma membrane is to maintain the necessary difference in composition between two compartments by restricting or permitting the passage of materials through the membrane as a function of intracellular signaling.[0004]Each cell has a resting membrane potential originating from the so-called “separation of charge” across th...

AI Technical Summary

Problems solved by technology

Transport of ions across the membrane through ion channels will lead to disturbance of the existing equilibrium of ion concentrations on both sides of membrane and, thus, to changes of electrical properties of the cell.

In humans, inherited or induced changes in ion channel function could result in serious complications to health.

Voltage-gated calcium ion channels are involved in numerous cellular functions, and their role in generating a defined disease phenotype is complex.

Most notably, mutations of the HERG potassium ion channels expressed in cardiac tissues or pharmacological blockage of HERG channels cause heart disease (long Q-T syndrome), which leads to increased risk of ventricular tachycardia and sudden death.

Several drugs affect these channels and can lead to life threatening cardiac arrhythmias.

Although this technique allows detailed biophysical characterization of ion channel activation, inactivation, gating, ion selectivity, and drug interactions, throughput is quite low and ease-of-use of patch-clamp instrumentation is generally unsatisfactory for effective mass screening.

The search for molecules that modulate ion channel function has been hindered by the lack of direct electrical measurements in HTS formats.

Real-time measurements of transmembrane potential kinetics that accurately reflect ion channel activity are fundamental to cell physiology, but are difficult to measure in existing HTS format methods.

Competition-binding assays, although successfully used for other target classes, often fail to identify ligands that modulate specific ion channel states.

Unfortunately binding assays only detect binding of compounds to ion channels and do not reveal changes in target function, such as modulation of ion channel kinetics.

However, utilization of DiBAC4(3) has several limitations, including slow kinetics (in the seconds to minutes range) and fluctuations in response to changes in temperature and concentration of the dye.

In addition, screening experiments using bisoxonol dyes require multi-step procedures and take 30-60 min for dye loading, potentially compromising the fidelity and reducing throughput of DiBAC4(3)-based screening assays.

Whole-cell patch-clamp, however, is not suitable for initial high throughput screening.

Although very powerful, this technique is labor-intensive and, therefore, limited to few data point measurements per day.

Currently used fluorescent voltage sensor dyes, which respond to potential-dependent accumulation and redistribution across the cellular membrane, are limited to steady-state assays of membrane potential.

Thus, redistribution-based voltage sensor dyes are prone to false-negatives.

In addition, compound-voltage dye interactions can show high false-positive rates.

The temporal resolution in fluorescence-based ion-channel assays using voltage-sensor dyes reduces the accessible kinetic range relative to patch-clamp-based ion channel assays.

A significant disadvantage of calcium dye-based ion-channel assays is their slow kinetic resolution of changes of intracellular calcium concentration, due to uncontrolled or unpredictable cellular processes.

This can interfere with assay results.

However, until now biological and biotechnological applications of nanocrystals have been mostly limited to their use as biomarkers rather than as detectors of biological processes.

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