Methods of registering trans-membrane electric potentials

Details Methods of registering trans-membrane electric potentials Methods of registering trans-membrane electric potentials

2000-03-23

2001-09-11

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S004000, C435S006120, C435S091100, C435S091200, C436S086000, C436S087000, C436S088000, C436S164000, C436S166000, C436S172000, C436S175000, C436S177000, C422S067000

Reexamination Certificate

active

06287758

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of cell biology, more particularly to measuring electric potentials across cell plasma and mitochondrial membranes.
BACKGROUND OF THE INVENTION
The existence of an electric potential across cell membranes, such as plasma membranes or the mitochondria, is a major factor in proper cell functioning. Excitatory cells, including neurons or muscle cells, actively separate negatively charged molecules inside the cell from positively charged ones in the space outside the membrane. This charge distribution maintains a steady-state homeostasis trans-membrane electric potential that is characteristic for resting cells. Upon cell activation, either with an electric field or with specific signal transducing molecules, the resting membrane potential changes. This change causes a cell reaction, which leads to, for example, neuronal signal propagation or muscular contraction.
The ability to monitor the cell membrane potential in single cells or in cell populations is important for understanding both the intricate molecular mechanisms underlying cell functioning, as well as for drug development. Two main approaches have been developed to monitor cell membrane potentials: direct electrical measurements with micro-electrodes and indirect measurement of membrane potential by following the redistribution of specially developed lipophilic ions labeled with either an isotope or a fluorescent moiety.
Both membrane permeable probes and non-membrane-permeable probes are used to monitor cell membrane potentials. Permeable fluorescent probes usually have high sensitivity to membrane potential changes but are very slow to respond to these changes. Non-permeable probes usually react quickly to membrane potential changes but have very low sensitivity. The main disadvantage of these probes is that their fluorescence intensity changes upon membrane depolarization or hyperpolarization. An approach based on fluorescence intensity can be confounded by variations in dye loading, cell density, or variability in excitation intensity. Also, it can be misleading when one considers the use of compounds with inherent spectral characteristics that interfere with the fluorescence of a probe.
As an example of membrane permeable probes, the class of negatively charged oxonols is best suited for measuring plasma membrane potentials because they are excluded from entering mitochondria due to the high negative charge inside the mitochondria. The oxonols are represented by a family of structures with the following general formulas (1 and 2):
In these formulas, Q is either O or S and each R is independently chosen from an alkyl or aryl group of 1 to 20 carbon atoms. These compounds are commercially available through various sources including Molecular Probes, Inc. (Eugene, Oreg.).
The bis-isoxazolone oxonols of Formula 1, namely Oxonol V and Oxonol VI (FIG.
2
), have been used for measuring membrane potentials mainly by absorption rather than fluorescence [Salvador et al.,
J. Biol. Chem.
273:18230-18234, 1998; Smith et al.,
J. Memb. Biol.
46:255-282, 1979] (incorporated herein by reference). The bis-barbituric acid oxonols of formula 2 are used for monitoring predominantly plasma membrane by changes in fluorescence intensity upon cell membrane depolarization or hyperpolarization [Epps et al.,
Chem. Phys. Lipids
69:137-150, 1994; Brauner et al.,
Biochim. Biophys. Acta.
771:208-216, 1984] (incorporated herein by reference).
Another class of widely used membrane permeable dyes comprises carbocyanine derivatives of the following general Formula 3:
In this formula, Q is O, S or C(CH
3
)
2
and each R is independently an alkyl group of 1 to 20 carbon atoms. These dyes, Indo- (DiI), thia- (DiS) and oxa- (DiO) carbocyanines with R ranging from one to seven carbon atoms, were the first potential sensitive probes developed [Sims et al.,
Biochemistry
13:3315-3330, 1974] (incorporated herein by reference). These molecules, being positively charged, concentrate on the surface and inside the plasma membrane [Cabrini et al.,
J. Membr. Biol.
92:171-182, 1986] (incorporated herein by reference) and mitochondria [Bunting et al., supra.] (incorporated herein by reference), where they aggregate with subsequent quenching of the fluorescence. This fluorescence intensity decrease is caused by potential-dependent binding of the molecules onto the membrane and aggregate formation [Guillet et al., supra.] (incorporated herein by reference). The main parameter used to monitor membrane potential in intact cells is the fluorescence intensity of dye in water, non-quenched phase of the dye. In this case it makes it impossible to distinguish between membrane potential changes of plasma membrane from that of mitochondria since an overall intensity for the cells is measured.
Alternative approaches have been developed [U.S. Pat. No. 5,661,035; Gonzalez et al.,
Chem. Biol.
4:269-277, 1997; Gonzalez et al.,
Biophys. J.
69:1272-1280, 1995] (incorporated herein by reference) that utilize Fluorescence Resonance Energy Transfer (FRET) phenomenon that can register membrane potentials in a ratiometric manner. These approaches and the matter compositions offered to practice FRET can elicit fast responses with high sensitivity to membrane potential changes. Unfortunately, these approaches and compositions are likely to cause artificial alterations in membrane structure and as a consequence, in cell functional behavior due to the unnatural incorporation of the highly hydrophobic dyes into the cell membrane. Additionally, use of fluorescently labeled lectin (WGA) as an affinity anchor for the second energy transfer counterpart reagent, can provoke cell functional responses by itself.
SUMMARY OF THE INVENTION
The present invention provides improved compounds and methods for measuring cell membrane electrical potential. In particular, the present invention uses donor and acceptor molecules to provide a fluorescent signal that is both rapid and sensitive to changes in membrane potential.
One embodiment of the present invention is a method for identifying compounds having biological activity comprising combining living cells with a first membrane penetrative dye and with a second membrane penetrative dye to form a test cell mixture; combining the test cell mixture with a test compound to form a test cell/compound mixture; placing said test cell/compound mixture into a detection zone; and measuring a cellular response in said test cell. Preferably, the biological activity is an initiation of the cellular response. Advantageously, the biological activity is a block of the cellular response In one aspect of this preferred embodiment, the cellular response is measured by a change in plasma membrane electric potential of the cells. Preferably, the cells are in a suspension. Alternatively, the cells are adhered to a substrate. In another aspect of this preferred embodiment, the substrate is beads, a microscope slide or a well of a multi-well plate. Preferably, the test compound is in solution. In one aspect of this preferred embodiment, the test compound solution comprises a standard compound having known biological effect. Preferably, the standard compound is an ion channel opener, ion channel blocker, ion transporter blocker or ion pump blockers. Advantageously, the plasma membrane electric potential is measured by fluorescence energy transfer between the first membrane penetrative dye and the second membrane penetrative dye. Preferably, the first dye is a fluorescent lipophilic anion having a characteristic excitation maximum between about 300 nm and 800 nm. Advantageously, the second dye is a fluorescent lipophilic anionic molecule having a characteristic excitation maximum which overlaps with the emission spectrum of the first dye. In one aspect of this preferred embodiment, the second dye has a characteristic excitation maximum of between about 220 nm and 700 nm. Preferably, the test cell/compound mixture is contacted with l

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