Voltage-dependent translocation of R18 and DiI across lipid bilayers leads to fluorescence changes.

We show that the lipophilic, cationic fluorescent dyes R18 and Dil translocate from one monolayer of a phospholipid bilayer membrane to the other in a concentration and voltage-dependent manner. When the probes were incorporated into voltage-clamped planar membranes and potentials were applied, displacement currents resulted. The charged probes sensed a large fraction of the applied field. When these probes were added to only one monolayer, displacement currents were symmetrical around 0 mV, indicating that the probes distributed equally between the two monolayers. Charge translocation required that the bilayer be fluid. When membranes were in a condensed gel phase, displacement currents were not observed; raising the temperature to above the gel-liquid crystalline transition restored the currents. Translocation of R18 was also shown by fluorescence measurements. When R18 was in the bilayer at high, self-quenching concentrations, voltage pulses led to voltage-dependent fluorescence changes. The kinetics of the fluorescence changes and charge translocations correlated. Adding the quencher I- to one aqueous phase caused fluorescence to decrease or increase when voltage moved R18 toward or away from the quencher at low, nonquenching concentrations of R18. In contrast to R18, Dil incorporated into bilayers was a carrier fo I-, and hence I- altered Dil currents. Voltage-driven translocations allow R18 and Dil to be used to probe membrane potential changes.     

Voltage-dependent conductance for alamethicin in phospholipid vesicles. A test for the mechanism of gating.

The ion currents induced by alamethicin were investigated in unilamellar vesicles using electron paramagnetic resonance probe techniques. The peptide induced currents were examined as a function of the membrane bound peptide concentration, and as a function of the transmembrane electrical potential. Because of the favorable partitioning of alamethicin to membranes and the large membrane area to aqueous volume in vesicle suspensions, these measurements could be carried out under conditions where all the alamethicin was membrane bound. Over the concentration range examined, the peptide induced conductances increased approximately with the fourth power of the membrane bound peptide concentration, indicating a channel molecularity of four. When the alamethicin induced currents were examined as a function of voltage, they exhibited a superlinear behavior similar to that seen in planar bilayers. Evidence for the voltage-dependent conduction of alamethicin was also observed in the time dependence of vesicle depolarization. These observations indicate that the voltage-dependent behavior of alamethicin can occur in the absence of a voltage-dependent phase partitioning. That is, a voltage-dependent conformational rearrangement for membrane bound alamethicin leads to a voltage-dependent activity.     

Effect of Voltage Sensitive Fluorescent Proteins on Neuronal Excitability

Fluorescent protein voltage sensors are recombinant proteins that are designed as genetically encoded cellular probes of membrane potential using mechanisms of voltage-dependent modulation of fluorescence. Several such proteins, including VSFP2.3 and VSFP3.1, were recently reported with reliable function in mammalian cells. They were designed as molecular fusions of the voltage sensor of Ciona intestinalis voltage sensor containing phosphatase with a fluorescence reporter domain. Expression of these proteins in cell membranes is accompanied by additional dynamic membrane capacitance, or “sensing capacitance”, with feedback effect on the native electro-responsiveness of targeted cells. We used recordings of sensing currents and fluorescence responses of VSFP2.3 and of VSFP3.1 to derive kinetic models of the voltage-dependent signaling of these proteins. Using computational neuron simulations, we quantitatively investigated the perturbing effects of sensing capacitance on the input/output relationship in two central neuron models, a cerebellar Purkinje and a layer 5 pyramidal neuron. Probe-induced sensing capacitance manifested as time shifts of action potentials and increased synaptic input thresholds for somatic action potential initiation with linear dependence on the membrane density of the probe. Whereas the fluorescence signal/noise grows with the square root of the surface density of the probe, the growth of sensing capacitance is linear. We analyzed the trade-off between minimization of sensing capacitance and signal/noise of the optical read-out depending on kinetic properties and cellular distribution of the probe. The simulation results suggest ways to reduce capacitive effects at a given level of signal/noise. Yet, the simulations indicate that significant improvement of existing probes will still be required to report action potentials in individual neurons in mammalian brain tissue in single trials.     

Dual-wavelength ratiometric fluorescence measurements of membrane potential

This work shows that the voltage across membranes in two very different preparations, lipid vesicles in suspension and individual HeLa cells under a microscope, is linearly related to the ratio of fluorescence excited from the two wings of the absorption spectrum of a voltage-sensitive dye. The dye di-4-ANEPPS [1-(3-sulfonatopropyl)-4-[beta-[2-(di-n-butylamino)-6-naphthyl] vin yl]pyridinium betaine] is well characterized from earlier investigations and responds via a rapid (less than millisecond) spectral shift to membrane potential changes. The resultant small change in fluorescence intensity monitored at a single wavelength is useful for measurements of temporally well-defined voltage transients such as action potentials. The dual-wavelength approach described in this work extends the usefulness of this fast potentiometric dye by filtering out complex or artifactual changes in fluorescence intensity and providing a voltage-dependent signal that is internally standardized. Thus, rapid measurements of membrane potential are made possible in nonexcitable cells.     

Measurement of transmembrane potentials in Rhodospirillum rubrum chromatophores with an oxacarbocyanine dye

The fluorescent dye 3,3-dipentyloxacarbocyanine (OCC) can be used as a fluorescence probe to measure transmembrane potentials across Rhodospirillum ruburm chromatophore membranes. A reversible fluorescence increase is observed in the light which is sensitive to inhibitors, permeable ions and uncouplers.Partial interchangeability between the electrical potential and the proton concentration gradient has been demonstrated by measurement of the fluorescence increase with OCC and the fluorescence quenching with 9-aminoacridine.OCC fluorescence changes can be induced also in the dark by injection of permeable salts and by rapid pH changes presumably indicating diffusion potentials. Using salt-induced diffusion potentials for calibrating the light signals and with several assumptions, the light-induced potentials were estimated as 170 mV for the maximal signal and 90–110 mV at the steady state.OCC has been shown to apparently increase the electrical conductivity of the chromatophore membrane, a fact which may be relevant to the mechanism of action of this probe.A red shift in the OCC absorption spectrum occurs when mixed with chromatophores, with a difference spectrum maximum at 495 nm. The absorption changes at 495 nm taking place in the light are similar in kinetics to the fluorescence changes. The absorbance spectrum of OCC in organic solvents is red shifted and the extent of the shift depends on the hydrophobicity of the medium. The difference spectrum compared to water in sec-butyl $\text{acetate}\text{n-}\text{hexane}$ (3 : 1, v/v) with a dipole moment of 5 was nearly identical to that of chromatophore-associated dye.The uncoupling properties of OCC at high concentrations and some difficulties in calibration limit the usefulness of this probe for quantitative measurements of transmembrane potentials.     

Tubulin-blocked state of VDAC studied by polymer and ATP partitioning

Recently reported functional interaction between voltage-dependent anion channel of the outer mitochondrial membrane, VDAC, and dimeric tubulin is observed as a reversible channel blockage. Using partitioning of poly-(ethylene glycol)s of different molecular weights and reversal potential measurements, we probe the size and ion selectivity of the fully open and tubulin-blocked states of VDAC reconstituted into planar lipid bilayers. While the effective radius of the channel decreases by only a factor of 1.34±0.15, the selectivity reverses from initially anionic to cationic. Directly measuring ATP partitioning we demonstrate that these changes prohibit ATP from entering the channel in its tubulin-blocked state.     

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.     

Single cell measurement of micro-viscosity by ratio imaging of fluorescence of styrylpyridinium probe

In aqueous solution, compounds containing the styrylpyridinium group showed dual fluorescence, in which excitation at either 469 or 360 nm each produced an emission band around 600 nm. The ratio of fluorescence intensities of the two bands (R = I469/I360) was sensitive to local viscosity. The N-carboxymethyl butyl ester of DMASP was found to be able to irreversibly load into a living cell; presumably by hydrolysis involving cellular lipases it was transformed to a membrane-impermeable fluorescent carboxylate. A map of the ratio, R, from a single cell was generated using fluorescence imaging microscopy with a spectrofluorimeter in dual-excitation single-emission mode. After calibrating the ratio for the probe in water/glycerol solutions, the intracellular viscosities were obtained for a single cell of smooth muscle of a rat embryonic thoracic aorta. The intracellular viscosity is differentiated inside the cell and the obtained values 18-7 cP obey all the values reported by other laboratories. Fluorescence emission of the probe (500-650 nm) is in a very favourable region for its use with visible fluorescence microscopy, without interferences from cell or tissue auto-fluorescence. The results present ability to detect and follow small changes in the ratio of fluorescence intensities, and apparently of the micro-viscosity.     

Oxonol VI as an optical indicator for membrane potentials in lipid vesicles

Experiments with large unilamellar dioleoylphosphatidylcholine vesicles were carried out in order to study the effect of membrane potential on the fluorescence of Oxonol VI. A partition equilibrium of dye between membrane and water was found to exist with a partition coefficient gamma identical to c lipid/c water of about 19,000 (at zero voltage). In the presence of an inside-positive membrane potential, the negatively charged dye accumulates in the intravesicular aqueous space according to a Nernst equilibrium. This leads to an increased adsorption of dye to the inner lipid monolayer and to a concomitant increase of fluorescence. The fluorescence change can be calibrated as a function of transmembrane voltage by generating a potassium diffusion potential in the presence of valinomycin. The intrinsic fluorescence of the membrane-bound dye is not affected by voltage; the whole influence of voltage on the fluorescence results from voltage-dependent partitioning of the dye between water and membrane. The voltage dependence of the apparent partition coefficient can be quantitatively described by a three-capacitor model in which the dye is assumed to bind to adsorption planes located on the hydrocarbon side of the membrane/solution interface. Oxonol VI was found to be suitable for detecting changes of membrane potential associated with the activity of the (Na+ + K+)-ATPase in reconstituted vesicles. When ATP is added to the external medium, pump molecules with the ATP-binding side facing outward become activated; this results in a translocation of net positive charge towards the vesicle interior. Under this condition, fluorescence changes corresponding to (inside-positive) potentials of up to 150-200 mV are observed. After the build-up of the membrane potential, a quasi-stationary state is reached in which the pump current is compensated by a back-flow of charge through passive conductance pathways.     

Fast and slow voltage sensor movements in HERG potassium channels

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH₂-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.     

Structural Implications of Fluorescence Quenching in the Shaker K+ Channel

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213–216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127–1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.     

Overview of the voltage-gated sodium channel family

Selective permeation of sodium ions through voltage-dependent sodium channels is fundamental to the generation of action potentials in excitable cells such as neurons. These channels are large integral membrane proteins and are encoded by at least ten genes in mammals. The different sodium channels have remarkably similar functional properties, but small changes in sodium-channel function are biologically relevant, as underscored by mutations that cause several human diseases of hyperexcitability.     

Very high frequency electron paramagnetic resonance of 2,2,6,6-tetramethyl-1-piperidinyloxy in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine liposomes: partitioning and molecular dynamics.

Partitioning and molecular dynamics of 2,2,6,6,-tetramethylpiperedine-1-oxyl (TEMPO) nitroxide radicals in large unilamellar liposomes (LUV) composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine were investigated by using very high frequency electron paramagnetic resonance (EPR) spectroscopy. Experiments carried out at a microwave frequency of 94.3 GHz completely resolved the TEMPO EPR spectrum in the aqueous and hydrocarbon phases. An accurate computer simulation method combined with Levenberg-Marquardt optimization was used to analyze the TEMPO EPR spectra in both phases. Spectral parameters extracted from the simulations gave the actual partitioning of the TEMPO probe between the LUV hydrocarbon and aqueous phases and allowed analysis of picosecond rotational dynamics of the probe in the LUV hydrocarbon phase. In very high frequency EPR experiments, phase transitions in the LUV-TEMPO system were observed as sharp changes in both partitioning and rotational correlation times of the TEMPO probe. The phase transition temperatures (40.5 +/- 0.2 and 32.7 +/- 0.5 degrees C) are in agreement with previously reported differential scanning microcalorimetry data. Spectral line widths were analyzed by using existing theoretical expressions for motionally narrowed nitroxide spectra. It was found that the motion of the small, nearly spherical, TEMPO probe can be well described by anisotropic Brownian diffusion in isotropic media and is not restricted by the much larger hydrocarbon chains existing in ripple structure (P beta') or fluid bilayer structure (L alpha) phases.     

Potential-dependent passive transport of calcium in myocardium sarcolemma vesicles

The effect of membrane potential on passive Ca2+ transport in isolated cardiac sarcolemmal vesicles was investigated. The membrane potentials were induced by creating potassium gradients across the vesicular membranes in the presence of valinomycin. The fluorescence changes in the voltage-sensitive dye, dis-C3(5), were consistent with the induction of potassium equilibrium potentials. The rate of 45Ca2+ efflux from inside-out vesicles was considerably greater at 0 than at -80 or +55 mV; prepolarization of the membrane to +90 mV did not enhance the 45Ca2+ efflux upon subsequent depolarization. The voltage-dependent 45Ca2+ efflux increased with a rise in internal Ca2+ concentration and exhibited a saturation effect. Furthermore, evaluation of the rate of 45Ca2+ efflux over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. It is concluded that the voltage-dependent Ca2+ efflux from the vesicles occurs via Ca2+-channels.     

Local mobility in lipid domains of supported bilayers characterized by atomic force microscopy and fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is used to examine mobility of labeled probes at specific sites in supported bilayers consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domains in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Those sites are mapped beforehand with simultaneous atomic force microscopy and submicron confocal fluorescence imaging, allowing characterization of probe partitioning between gel DPPC and disordered liquid DOPC domains with corresponding topography of domain structure. We thus examine the relative partitioning and mobility in gel and disordered liquid phases for headgroup- and tailgroup-labeled GM1 ganglioside probes and for headgroup- and tailgroup-labeled phospholipid probes. For the GM1 probes, large differences in mobility between fluid and gel domains are observed; whereas unexpected mobility is observed in submicron gel domains for the phospholipid probes. We attribute the latter to domain heterogeneities that could be induced by the probe. Furthermore, fits to the FCS data for the phospholipid probes in the DOPC fluid phase require two components (fast and slow). Although proximity to the glass substrate may be a factor, local distortion of the probe by the fluorophore could also be important. Overall, we observe nonideal aspects of phospholipid probe mobility and partitioning that may not be restricted to supported bilayers.     

Development of dansyl-modified oligonucleotide probes responding to structural changes in a duplex

We have synthesized a nonnucleoside amidite block of dansyl fluorophore to prepare dansyl-modified oligonucleotides (ONTs). The fluorescence intensities of dansyl-ONT specifically increased by the presence of adjacent guanosine residues but, significantly reduced in a dansyl-flipping duplex. These changes were caused by solvatochromism effect due to the number of guanine which is hydrophobic functional group and the external environment of dansyl group. The fluorescence intensities could be plotted as a function of the ONTs concentrations and the increase in the fluorescence was observed to equimolar concentrations of target DNA. This duplex exhibited higher melting temperature relative to the corresponding duplexes containing other base pairs. Similar changes in fluorescence could be detected upon hybridization with complementary RNAs. Thus, the dansyl-modified ONTs provide sequence specific fluorescent probe of DNA and RNA.     

Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy

The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.     

Membrane Disordering by Anesthetic Drugs: Relationship to Synaptosomal Sodium and Calcium Fluxes

The effects of membrane perturbants (ethanol, pentobarbital, chloroform, diethylether, phenytoin, cis-vaccenic acid methylester, and cis-vaccenoyl alcohol) on the lipid order of mouse brain synaptic plasma membranes (SPM) were tested by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. The compounds decreased the fluorescence polarization of both probes, indicating that they disordered the membrane lipids. The decrease in polarization was, however, greater for DPH than for TMA-DPH, suggesting a greater effect on the membrane core than on the membrane surface. The voltage-dependent uptake of 24Na and 45Ca was studied in isolated mouse brain synaptosomes as a measure of membrane function. All of the compounds inhibited sodium influx, and their potencies for decreasing sodium uptake and fluorescence polarization of DPH were linearly correlated (r = 0.91). The relationship between changes in sodium influx and TMA-DPH polarization was less consistent (r = 0.66). Synaptosomal calcium uptake was inhibited by most, but not all, of the perturbants, but this inhibition was poorly correlated with changes in fluorescence polarization of DPH (r = 0.36) or TMA-DPH (r = 0.26). These results indicate that the function of synaptic sodium channels is correlated with lipid order in the hydrophobic core of the membrane and that the inhibitory effects of intoxicant-anesthetic drugs on neuronal sodium fluxes may be the result of their capacity to disorder these lipids. In contrast, the effects of drugs on voltage-dependent calcium channels were not clearly related to the capacity of these agents to disorder membrane lipids.     

Kinetics of an individual transmembrane helix during bacteriorhodopsin folding

The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.     

Synchronous Ca(2+) oscillation emerges from voltage fluctuations of Ca(2+) stores

Synchronous Ca(2+) oscillation occurs in various cell types to regulate cellular functions. However, the mechanism for synchronization of Ca(2+) increases between cells remains unclear. Recently, synchronous oscillatory changes in the membrane potential of internal Ca(2+) stores were recorded using an organelle-specific voltage-sensitive dye [Yamashita et al. (2006) FEBS J273, 3585-3597], and an electrical coupling model of the synchronization of store potentials and Ca(2+) releases has been proposed [Yamashita (2006) FEBS Lett580, 4979-4983]. This model is based on capacitative coupling, by which transient voltage changes can be synchronized, but oscillatory slow potentials cannot be communicated. Another candidate mechanism is synchronization of action potentials and ensuing Ca(2+) influx through voltage-dependent Ca channels. The present study addresses the question of whether Ca(2+) increases are synchronized by action potentials, and how oscillatory store potentials are synchronized across the cells. Electrophysiological and Ca(2+)-sensitive fluorescence measurements in early embryonic chick retina showed that synchronous Ca(2+) oscillation was caused by releases of Ca(2+) from Ca(2+) stores without any evidence of action potentials in retinal neuroepithelial cells or newborn neurons. High-speed fluorescence measurement of store membrane potential surprisingly revealed that the synchronous oscillatory changes in the store potential were periodic repeats of a burst of high-frequency voltage fluctuations. The burst coincided with a Ca(2+) increase. The present study suggests that synchronization of Ca(2+) release is mediated by the high-frequency fluctuation in the store potential. Close apposition of the store membrane and plasma membrane in an epithelial structure would allow capacitative coupling across the cells.