Comparative study of membrane potential-sensitive fluorescent probes and their use in ion channel screening assays

In this study, the authors compared and evaluated 4 membrane potential probes in the same cellular assay: the oxonol dye DiBAC(4)(3), the FLIPR membrane potential (FMP) dye (Molecular Devices), and 2 novel fluorescence resonance energy transfer (FRET) dye systems from PanVera [CC2-DMPE/DiSBAC(2)(3)] and Axiom [DiSBAC(1)(3)/DiSBAC(1)(5)]. The kinetic parameters of each membrane probe were investigated in RBL-2H3 cells expressing an endogenous inward rectifier potassium channel (IRK1). The FMP dye presented the highest signal over background ratio whereas the FRET dyes from PanVera gave the fastest response. The determination of IC(50) values for 8 different channel modulators indicated a good correlation between the 4 membrane probe systems. The compound-dye interaction was evaluated in the presence of compounds at 10 muM and clearly indicated no effect on the FMP or the PanVera donor dye, whereas some major interference with the oxonol probes was observed. Using a cell permeabilization assay in the presence of gramicidin, the authors concluded that the FRET dyes from PanVera and the FMP dye are unable to measure the gramicidin-induced cell membrane hyperpolarizations. The 4 dye systems were investigated under high-throughput screening (HTS) conditions, and their respective Z' parameter was determined. The characteristics of each dye system and its potential use in HTS assays is discussed.     

The use of voltage-sensitive dyes to monitor signal-induced changes in membrane potential-ABA triggered membrane depolarization in guard cells

The plant membrane potential reports on the activity of electrogenic plasma membrane transport processes. The membrane potential is widely used to report for early events associated with changes in light regime, hormone action or pathogen attacks. The membrane potentials of guard cells can be precisely measured with microelectrodes, but this technique is not well suited for rapid screens with large sample numbers. To provide the basis for large-scale membrane potential recordings, we took advantage of voltage-sensitive dyes. Using the fluorescent dyes bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC(4)(3)) and the FLIPR Membrane Potential Assay Kit (FMP) dye we followed changes in the membrane potential in guard cells and vacuoles. Based on the fluorescence of DiBAC(4)(3) a method was established for quantification of the membrane potential in guard cell protoplasts which should be considered as an excellent system for high-throughput screening of plant cells. In the absence of abscisic acid (ABA), one-third of the guard cell protoplast population spontaneously oscillated for periods of 5-6 min. Upon application of ABA the hyperpolarized fraction ( approximately 50%) of the guard cell protoplast population depolarized within a few minutes. Membrane potential oscillations were terminated by ABA. Oscillations and ABA responses were found in cell populations with active anion channels. Thus time- and voltage-dependent anion channels likely represent the ABA-sensitive conductance and part of the membrane potential oscillator. The suitability of membrane potential dyes was tested on vacuoles, too. Dye-based vacuolar membrane polarization was monitored upon ATP exposure. We conclude that voltage-sensitive dyes provide an excellent tool for the study of changes in the membrane potential in vacuole as well as guard cell populations.     

A microfluidic device for measuring cellular membrane potential

Recent developments in microfluidics have enabled the design of a lab-on-a-chip system capable of measuring cellular membrane potential. The chip accesses liquid samples sequentially by sipping from a microplate through a capillary, mixes the samples with cells flowing through a microchannel, contacts the cells with potential-sensitive dyes, and reads out cellular responses using fluorescence detection. The rate of cellular uptake of membrane-permeable, ionic fluorophores by THP-1 cells was found to depend strongly on membrane potential. The ratio of the fluorescence of the anionic dye DiBAC(4)(3) and the cationic dye Syto 62 taken up by cells was found to double for every 33 mV change in membrane potential. The utility of this approach was demonstrated by assaying ion channel activity in human T lymphocytes. Because of the high sensitivity, low cellular and reagent consumption, and high data quality obtained with the microfluidic device, the lab-on-a-chip system should be widely applicable in high-throughput screening and functional genomics studies.     

Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons.

An experimental method has been established to measure the electric properties of a cell membrane by combination of patch clamp and dual-wavelength ratio imaging of a fluorescent potentiometric dye, 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl ]pyridinium betaine (di-8-ANEPPS). Pairs of fluorescence images from the dye-stained membrane of neuroblastoma N1E-115 cells excited at two wavelengths were initially obtained to calculate ratio images corresponding to the resting transmembrane potential. Subsequently, a whole-cell patch was established and the membrane potential clamped to levels varying from -100 to +60 mV; at each voltage, a pair of dual-wavelength images were acquired to develop a calibration of the fluorescence ratio. Using this method, the resting potentials could accurately be measured showing that the differentiated cells were 17 mV more polarized than undifferentiated cells. The combination of electrical and optical methods can also follow changes in other membrane electric properties, such as dipole potential, and thus permit a detailed analysis of the membrane electrical properties underlying the voltage regulation of ion channels.     

Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers

Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC(4)(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC(4)(5). The peak DeltaF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC(4)(5) exhibit DeltaF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.     

Role of chloride in constriction of descending vasa recta by angiotensin II

We investigated the dependence of ANG II (10(-8) M)-induced constriction of outer medullary descending vasa recta (OMDVR) on membrane potential (Psim) and chloride ion. ANG II depolarized OMDVR, as measured by fully loading them with the voltage-sensitive dye bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC(4)(3)] or selectively loading their pericytes. ANG II was also observed to depolarize pericytes from a resting value of -55.6 +/- 2.6 to -26.2 +/- 5.4 mV when measured with gramicidin D-perforated patches. When measured with DiBAC(4)(3) in unstimulated vessels, neither changing extracellular Cl(-) concentration ([Cl(-)]) nor exposure to the chloride channel blocker indanyloxyacetic acid 94 (IAA-94; 30 microM) affected Psim. In contrast, IAA-94 repolarized OMDVR pretreated with ANG II. Neither IAA-94 (30 microM) nor niflumic acid (30 microM, 1 mM) affected the vasoactivity of unstimulated OMDVR, whereas both dilated ANG II-preconstricted vessels. Reduction of extracellular [Cl(-)] from 150 to 30 meq/l enhanced ANG II-induced constriction. Finally, we identified a Cl(-) channel in OMDVR pericytes that is activated by ANG II or by excision into extracellular buffer. We conclude that constriction of OMDVR by ANG II involves pericyte depolarization due, in part, to increased activity of chloride channels.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy

The patch-clamp technique has enabled functional studies of single ion channels, but suffers limitations including lack of spatial information and inability to independently monitor currents from more than one channel. Here, we describe the use of total internal reflection fluorescence microscopy as an alternative, noninvasive approach to optically monitor the activity and localization of multiple Ca(2+)-permeable channels in the plasma membrane. Images of near-membrane Ca(2+) signals were obtained from >100 N-type channels expressed within restricted areas (80 x 80 micro m) of Xenopus oocytes, thereby permitting simultaneous resolution of their gating kinetics, voltage dependence, and localization. Moreover, this technique provided information inaccessible by electrophysiological means, demonstrating that N-type channels are immobile in the membrane, show a patchy distribution, and display diverse gating kinetics even among closely adjacent channels. Total internal reflection fluorescence microscopy holds great promise for single-channel recording of diverse voltage- and ligand-gated Ca(2+)-permeable channels in the membrane of neurons and other isolated or cultured cells, and has potential for high-throughput functional analysis of single channels.     

Ginsenosides regulate ligand-gated ion channels from the outside

Treatment with ginsenosides, the major active ingredients of Panax ginseng, produces a variety of physiological effects on the central and peripheral nervous systems. Ginsenosides inhibit various types of ligand-gated ion channel but it is not clear whether they act from within or outside the cell since they are somewhat membrane-permeable. In the present study, we used the Xenopus oocyte gene expression system to determine from which side of the cell membrane the ginsenoside Rg3 (Rg3), and M4, a ginsenoside metabolite, act to regulate ligand-gated ion channel activity. Ligand-gated ion currents were measured using the two-electrode voltage clamp technique. Rg3 and M4 inhibited 5-HT3A and a3b4 nACh receptor-mediated ion currents when present outside of the cell but not when injected intracellularly. We also examined the effect of these agents on oocytes expressing the gustatory cGMP-gated ion channel, which is known to have a cGMP binding site on the intracellular side of the plasma membrane and is only activated by cytosolic cGMP. Rg3 inhibited cGMP-gated ion currents when applied extracellularly or to an outside-out patch clamp, but not when injected into the cytosol or when using an excised inside-out patch clamp. These results indicate that Rg3 and M4 regulate ligand-gated ion channel activity from the extracellular side.     

Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K~ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage-sensitive channels. Single ATP-sensitive K (K-ATP) channel currents were recorded in acutely dissociated rat neo-cortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intra-cellular ATP, but also by membrane potential ( Vm) , and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and di-versity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activa-tion-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary con-ductance of about 80 pS in detail ( n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane poten     

"Optical patch-clamping": single-channel recording by imaging Ca2+ flux through individual muscle acetylcholine receptor channels

We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of alphabetagammadelta subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel "channel chip" representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.     

Monitoring of real changes of plasma membrane potential by diS-C3(3) fluorescence in yeast cell suspensions

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.     

Fluorescence imaging of electrically stimulated cells

Designing high-throughput screens for voltage-gated ion channels has been a tremendous challenge for the pharmaceutical industry because channel activity is dependent on the transmembrane voltage gradient, a stimulus unlike ligand binding to G-protein-coupled receptors or ligand-gated ion channels. To achieve an acceptable throughput, assays to screen for voltage-gated ion channel modulators that are employed today rely on pharmacological intervention to activate these channels. These interventions can introduce artifacts. Ideally, a high-throughput screen should not compromise physiological relevance. Hence, a more appropriate method would activate voltage-gated ion channels by altering plasma membrane potential directly, via electrical stimulation, while simultaneously recording the operation of the channel in populations of cells. The authors present preliminary results obtained from a device that is designed to supply precise and reproducible electrical stimuli to populations of cells. Changes in voltage-gated ion channel activity were monitored using a digital fluorescent microscope. The prototype electric field stimulation (EFS) device provided real-time analysis of cellular responsiveness to physiological and pharmacological stimuli. Voltage stimuli applied to SK-N-SH neuroblastoma cells cultured on the EFS device evoked membrane potential changes that were dependent on activation of voltage-gated sodium channels. Data obtained using digital fluorescence microscopy suggests suitability of this system for HTS.     

Descending vasa recta endothelium is an electrical syncytium

We examined gap junction coupling of descending vasa recta (DVR). DVR endothelial cells or pericytes were depolarized to record the associated capacitance transients. Virtually all endothelia and some pericytes exhibited prolonged transients lasting 10-30 ms. Carbenoxolone (100 microM) and 18beta-glycyrrhetinic acid (18betaGRA; 100 microM) markedly shortened the endothelial transients. Carbenoxolone and heptanol (2 mM) reduced the pericyte capacitance transients when they were prolonged. Lucifer yellow (LY; 2 mM) was dialyzed into the cytoplasm of endothelial cells and pericytes. LY spread diffusely along the endothelial monolayer, whereas in most pericytes, it was confined to a single cell. In some pericytes, complex patterns of LY spreading were observed. DVR cells were depolarized by voltage clamp as fluorescence of bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)] was monitored approximately 200 microm away. A 40-mV endothelial depolarization was accompanied by a 26.1 +/- 5.5-mV change in DiBAC(4)(3) fluorescence. DiBAC(4)(3) fluorescence did not change after 18betaGRA or when pericytes were depolarized. Similarly, propagated cytoplasmic Ca(2+) responses arising from mechanical perturbation of the DVR wall were attenuated by 18betaGRA or heptanol. Connexin (Cx) immunostaining showed predominant linear Cx40 and Cx43 in endothelia, whereas Cx37 stained smooth muscle actin-positive pericytes. We conclude that the DVR endothelium is an electrical syncytium and that gap junction coupling in DVR pericytes exists but is less pronounced.     

Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K+ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage sensitive channels. Single ATP-sensitive K⁺ (K-ATP) channel currents were recorded in acutely dissociated rat neocortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intracellular ATP, but also by membrane potential (V_m), and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and diversity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activation-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary conductance of about 80 pS in detail (n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane potentials. This property of the K-ATP channels may be related to their pathophysiological function.     

A real-time screening assay for GIRK1/4 channel blockers

The cardiac acetylcholine-activated K(+) channel (I(K,Ach)) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K(+) (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K(+) efflux through the GIRK1/4 channel (carbachol vs. control solution, Z' factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl(2) and enhanced by increasing the driving force for K(+) across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na(+) and K(+) channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 μM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF.     

State-dependent compound inhibition of Nav1.2 sodium channels using the FLIPR Vm dye: on-target and off-target effects of diverse pharmacological agents

Voltage-gated sodium channels (NaChs) are relevant targets for pain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (Vm) dyes in conjunction with a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus, the authors have developed a FLIPR Vm assay of rat Nav1.2 NaCh. Channels were opened by addition of veratridine, and Vm dye responses were measured. The IC50 values from various structural classes of compounds were compared to the resting state binding constant (Kr)and inactivated state binding constant (Ki)obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with Ki but not Kr. FLIPRIC50 values fell within 0.1-to 1.5-fold of EP Ki values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology.     

Imaging single-channel calcium microdomains

The Ca(2+) microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca(2+) signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca(2+) imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of 'optical patch-clamp recording' is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca(2+) ions.