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Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095

Correspondence to Julio L. Vergara: jvergara{at}mednet.ucla.edu

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
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 DiBAC₄(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 DiBAC₄(5). The peak F/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 DiBAC₄(5) exhibit F/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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
Nonradiative energy transfer (Föster energy transfer) is a valuable tool to measure intra- and intermolecular distances (within the 1–10-nm range) between donor and acceptor fluorophores (FRET pairs) (Stryer and Haugland, 1967; Clegg, 1995). The steep dependence of FRET on the distance between interacting fluorophores, and the fact that the thickness of biological membranes is within the effective range of FRET interaction for commonly used fluorophores, have been exploited in the design of photometric methods to measure transmembrane potential. Thus, FRET pairs between acceptor (donor) lipophilic mobile ions and donor (acceptor) fluorophores bound (or anchored) at either side of membrane have been reported (Gonzalez and Tsien, 1995, 1997). The voltage dependence of the FRET signals stems from the voltage-dependent translocation of a lipophilic ion between positions close to the inner and outer leaflet of the membrane, representing two energy minima (Gonzalez and Tsien, 1995, 1997; Chanda et al., 2005). Both the mobile and fixed components of voltage-sensing FRET pairs were initially envisaged as synthetic chemical compounds staining either side of the cell membrane (Gonzalez and Tsien, 1995, 1997). However, a recently described hybrid voltage-sensing system relies on the FRET interaction between recombinant farnesylated EGFP (EGFP-F) expressed at the surface membrane of neurons and cells in culture and the nonfluorescent absorber dipicrylamine (DPA) (Chanda et al., 2005). This method takes advantage of well-studied targeting mechanisms (through farnesylation signals) by which c-Ha-Ras proteins are anchored to the inner leaflet of the cell membrane (Aronheim et al., 1994; Moriyoshi et al., 1996; Zhang and Casey, 1996). A voltage-dependent optical signal is then generated by the resonance interaction between excited EGFP-F molecules anchored to the membrane, which act as donors, and DPA molecules that work as nonfluorescent mobile acceptors. In this case, the mechanism of energy transfer is the same as in FRET (hence the primary theoretical formulas can be used [Clegg, 1995; Siegel et al., 2000; Selvin, 2002]), but since the only detectable optical change is the quenching of the donor fluorescence by the acceptor, it is called QRET signal. We will adopt this nomenclature to distinguish this type of signal from those generated by two fluorophores in which FRET optical changes, "donor quenching" and "acceptor-sensitized emission," can be detected (Gonzalez and Tsien, 1995).

The hybrid QRET pair has some advantages over methods based on the use of synthetic fluorophores because the expression of the donor can be made cell type dependent, thus allowing for the recording of electrical activity from specific subpopulation of cells. Furthermore, the small size and small translocation time constant of DPA make the EGFP-F//DPA QRET pair capable of tracking rapid changes in membrane potential (frequency response >2 kHz) (Fernandez et al., 1983).

In the current report, we demonstrate that murine skeletal muscle fibers readily express EGFP-F, and the novel farnesylated cyan fluorescent protein (ECFP-F), in the surface and transverse tubular system (TTS) membranes. Further, we show that these anchored fluorescent proteins can be combined with DPA to dynamically probe changes in membrane potential arising at this prominent muscle membrane compartment. We also report a novel hybrid FRET system to follow changes in membrane potential based on the use of EGFP-F as a stationary donor and the dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC₄(5)) as a mobile acceptor. Finally, the voltage-sensing characteristics of FRET-based systems were analyzed in the light of the predictions from a radial cable model of the muscle fiber (Ashcroft et al., 1985; Kim and Vergara, 1998b) in which a two-state model (Fernandez et al., 1983) of the voltage-dependent translocation of mobile ions was incorporated in the surface and TTS membranes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
Biological Preparation
Animal handling and protocols were performed according to the guidelines laid down by the UCLA Animal Research Committee. Single fibers from flexor digitorum brevis (FDB) and interossei (IOS) muscles from the lower limb of 2–6-mo C57BL/10J mice were used. Intact fibers were obtained by enzymatic dissociation and transferred to an optical chamber, which was mounted on the stage of an inverted microscope (IX-71, Olympus) equipped with an epifluorescence attachment (DiFranco et al., 2005; Woods et al., 2005).

Plasmids for Recombinant Farnesylable and Soluble EGFP and ECFP
The pEGFP-F vector, encoding for recombinant EGFP containing a 20–amino acid farnesylation signal from the c-Ha-Ras protein, was commercially available (CLONTECH Laboratories, Inc.). It was amplified and used for in vivo electroporation without further modification. A novel plasmid encoding for the same farnesylation signal, but attached to ECFP (ECFP-F) was constructed by us by replacing the NheI-BsrGI fragment of pEGFP-F with the NheI-BsrGI fragment from pECFP-C1 (CLONTECH Laboratories, Inc.). Plasmids encoding for EGFP and ECFP N-tagged with a 6-His sequence (pEGFPN1-histag and pECFPN1-histag, respectively) were engineered from pECFP-N1 and pEGFP-N1 (CLONTECH Laboratories, Inc.).

Transfection and Expression of pECFP-F and pEGFP-F in Muscle
FDB and IOS muscles were transfected in vivo using an electroporation protocol similar to that reported elsewhere (DiFranco et al., 2006). In brief, as illustrated in Fig. 1, 5 µl of 2 mg/ml hyaluronidase (type IV-S from bovine testes, Sigma-Aldrich) dissolved in medical grade sterile saline was injected subcutaneously in the lower limb footpads using 33-gauge needles (Hamilton). 1 h later, 25 µg pDNA (5 µg/µl in sterile buffer TE) was injected the same way. After 10 min, the muscles were electroporated by applying 20 pulses (1Hz, 20 ms duration each) between two subcutaneous electrodes (see Fig. 1). The amplitude of the pulses was adjusted to apply a field of 100 V/cm. The electrodes consisted of disposable sterile gold-plated acupuncture needles 0.2 x 25 mm (Lhasa OMS, Inc.) which were connected via microclips to a medical grade stimulator (Grass S88, Grass-Telefactor). Injections and electroporation were performed under anesthesia (3% isoflurane in O₂, 2 liters/min). Transfected muscles were used for fiber isolation 2–10 d after electroporation. The efficiency of expression and intracellular targeting of EGFP-F and ECFP-F in the muscle fibers were verified in two-photon laser scanning microscopy (TPLSM) images acquired with a 20x, 0.95 NA (Olympus XLUMPLANFL) water immersion objective. The TPLSM system was based on an upright microscope (BX51WI, Olympus) equipped with a tunable wavelength Chameleon Ti/Sapphire laser (Coherent) and a Radiance 2000 Scanning Head (Bio-Rad Laboratories). Both ECFP-F and EGFP-F were excited at 890 nm and the fluorescence detected through 450//460-500 and 500//510-560 dichroic//band-pass filter combinations, respectively. The precise targeting of farnesylated fluorescent proteins was determined in muscles transfected with pECFP-F and subsequently stained with di-8-ANEPPS using TPLSM. Both ECFP-F and di-8-ANEPPS were excited at 890 nm and their emission was separated with a 550-nm dichroic mirror, and filtered with 460–500 and 580–630-nm band-pass filters, respectively. Two photon images were analyzed using commercial (LaserSharp 2000MP, Bio-Rad Laboratories, Carl Zeiss MicroImaging, Inc.) and/or public domain image analysis software packages (ImageJ). The images were finally rendered in 256 pseudocolor intensity levels using monochromatic pseudocolor scales that match the color of the respective fluorophores' emission.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Plasmid DNA transfection in murine foot muscles by in vivo electroporation. The schematic diagram indicates the sites where injection and electrode needles were placed in mice feet with respect to the positions of footpads and toes (plantar view). See text for a detailed explanation of the protocols.

Electrophysiology
A two-microelectrode amplifier (Dagan, TEV-200A) was used for stimulation and recording of electrical signals from isolated muscle fibers under both current (CC) and voltage-clamp (VC) configurations, as described elsewhere (Woods et al., 2004; DiFranco et al., 2005; Woods et al., 2005). To improve the frequency response of the voltage-clamp system, the microelectrodes were drawn to the largest tip compatible with fiber viability. Voltage microelectrodes were filled with either 1 M KCl (CC) or 2 M CsCl (VC) and had resistances 10 M; current electrodes were filled with either K (CC) or Cs (VC) based internal solutions (see Solutions) and had resistances of 15 M. The tips of the microelectrodes were placed 10 µm apart along the midline of the muscle fiber. Microelectrode capacitance was maximally compensated with a positive feedback circuit. In CC experiments the muscle fibers were bathed in Tyrode solution and the membrane potential was adjusted to –90 mV by injecting a steady current, typically <15 µA/cm² (Woods et al., 2004); action potentials (APs) were elicited with 0.5-ms current pulses. In VC experiments, the fibers were rendered electrically passive by replacing Tyrode with V-clamp external solution (see Solutions) and maintained at a –90 mV holding potential (HP). A 16-bit data acquisition board (National Instruments, PCI-MIO-16XE-10) under program control (Labview, National Instruments) was used to synchronously generate stimulus pulses and to acquire data. Total membrane capacitance was calculated by integration of current records subtracted from linear leak. Unless otherwise stated, voltage and current records were filtered at 5 kHz with 8-pole Bessel filters (Frequency Devices), and digitized at >30 kHz. Experiments were performed at room temperature (21°C).

Muscle Fiber Staining with Potentiometric Dyes and DPA
Staining of the muscle fibers with DPA (ammonium salt, City of Chemicals) and DiBAC₄(5) (Invitrogen) was performed in the recording chamber by transient perfusion (5–10 min) with dye-containing external solutions (5 µM) followed by extensive wash with dye-free solution. Solutions were made fresh by dilution from DMSO stocks (10–100 mM). Staining with di-8-ANEPPS was as previously described (DiFranco et al., 2005).

Optical Recordings
The optical arrangement for recording global fluorescence, QRET, and FRET signals is similar to that described elsewhere (DiFranco et al., 2005). Light from a Tungsten halogen lamp, filtered using the filter configurations described in Table I, was focused onto the fibers' medial plane to form an 15-µm diameter disc using a 100x, 1.4 NA oil immersion objective (Olympus). The fluorescence image of the disc was focused on a PIN photodiode (UV100, United Detector Technologies) connected to a current-to-voltage converter (Kim and Vergara, 1998a; Woods et al., 2004; DiFranco et al., 2005). Low pass–filtered (2–5 kHz) single sweep optical signals were acquired in synchrony with electrical signals (membrane voltage and current) and normalized to the prestimulus baseline (F/F). Movement artifacts in the optical signals were prevented by the use of 50 mM EGTA in the internal solutions (see below).

Spectral Properties of Dyes and Fluorescent Proteins
The absorbance spectra of DPA and DiBAC₄(5) dissolved in water and methanol, and of the fluorescent proteins in internal solution, were measured in an HP 8453 spectrophotometer. The fluorescence spectra of DiBAC₄(5), EGFP, and ECFP in solution were measured in a FluoroMax-3 Spectrofluorimeter (Horiba Jobin Yvon). The fluorescence spectra of EGFP-F and ECFP-F were measured in vivo from isolated fibers mounted in the fluorescence microscope described above but additionally equipped with a fiber optic–coupled spectrofluorimeter (EPP2000, StellarNet). The custom-made cube configuration for the acquisition of fluorescence spectra in vivo was 400–445//450//470-650.

The quantum yield () of EGFP and ECFP was determined using fluorescein as a standard reference of known following protocols described elsewhere (Williams et al., 1983; Magde et al., 2002); solutions of EGFP (ECFP) at four concentrations were prepared such that their absorbance values, measured at 460 (434) nm, ranged between 0.005 and 0.1. Fluorescein solutions in 100 mM NaOH with the same absorbance values were also prepared. Then, the fluorescence emission spectra of the proteins and fluorescein solutions (excited at the wavelengths above) were recorded. These fluorescence spectra were integrated (Berkeley Madonna, Oster & Macer) and the values of the integrals were plotted as a function of the corresponding absorbance for each solution. These data were fitted to straight lines and the slopes (m) were used to calculate the quantum yield according to the formula (Williams et al., 1983): _x = _Fl · (m_x/m_Fl) · (n²_x/n²_Fl), where x= EGFP or ECFP, and n is the refractive index of the solutions, which were measured (N = 3) using an Abbe-type refractometer (Bausch and Lomb). The values of n for NaOH and internal solutions were 1.336 ± 0.0006 and 1.342 ± 0.0007, respectively. The value of _Fl in 0.1 NaOH was assumed to be 0.92, as suggested from the literature consensus (Williams et al., 1983). For both proteins, measurements were made in two batches of four concentrations each, yielding values of 0.6 ± 0.12 and 0.62 ± 0.09 for EGFP and ECFP, respectively.

Data Analysis
Optical signals were analyzed and processed using a custom program written in Delphi (Borland Corporation) and Origin 7.5 (OriginLab). Kinetic parameters and amplitudes of optical signals were determined as described previously (DiFranco et al., 2002; Woods et al., 2004).

Current records obtained from voltage-clamped fibers under passive conditions were integrated using a custom written LabView (National Instruments) program and analyzed for best-fit parameters using least square fitting routines in Berkeley Madonna (Oster & Macey) and Origin 7.5. The on and off decay kinetics of the capacitive currents were fitted to the double exponential function

and integrated numerically to calculate the charge mobilized. I_SS is the steady-state current (leak) during the pulse and I₁ and ₁ and I₂ and ₂ are the amplitude and time constant of each transient component, respectively.

Solutions
The compositions of the solutions (in mM) were as follows: Tyrode: 150 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 10 dextrose, 10 MOPS, 0.5 Trolox, pH adjusted with NaOH; V-clamp external: 182 TEA-OH, 5 CsOH, 3.25 Ca(OH)₂, 5 dextrose, 15 MOPS, 0.0002 TTX, 0.002 verapamil, 1.0 9-ACA, 0.5 Trolox, pH adjusted with H₂SO₄; K-internal: 50 aspartic acid, 20 MOPS, 5 ATP-Tris, 5 MgCl₂, 50 EGTA, 5 di-Na phosphocreatine, 3 reduced glutathione, 5 dextrose, and 0.1 mg/ml phosphocreatine kinase, pH adjusted with KOH; Cs-internal: same as K-internal, but pH adjusted with CsOH. Osmolarity and pH of all the solutions were set at 300 mOsm and 7.4, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
Targeting of Farnesylated Fluorescent Proteins to External Muscle Membrane Systems
A prerequisite for the use EGFP-F or ECFP-F as effective immobile components of voltage-sensing QRET pairs is to demonstrate that they are specifically anchored at the desired external membrane compartments, which in the case of skeletal muscle are the TTS and sarcolemma membranes, while leaving the internal membrane compartments untagged. We used TPLSM to examine the specificity of the intracellular targeting of these proteins to the membrane systems of FDB and IOS muscle fibers. Fig. 2 A shows a TPLSM section of an FDB fiber obtained 2 d after transfection with pEGFP-F. As can be seen the fluorescence is restricted to the perimeter of the fiber and to bands oriented orthogonally to the long axis of the fiber, while it is mostly absent in the myoplasm and the nucleoplasm. The fluorescent bands are arranged in pairs, displaying a pattern reminiscent of that described previously in mammalian fibers stained with di-8-ANEPPS (DiFranco et al., 2005). It should be noted that EGFP-F is not targeted to the nuclear envelope, as evidenced by the absence of fluorescence in the internal (myoplasmic) face of the nuclei (Fig. 2 A, arrowheads). It is also observed that the poles of this organelle are associated with regions of high fluorescence intensity that extend in the subsarcolemmal region alongside the fiber (Fig. 2 A, arrows).

View larger version (66K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Membrane expression of EGFP-F and ECFP-F in FDB muscle fibers. (A) TPLSM image section obtained at the medial plane of a muscle fiber expressing EGFP-F. n denotes nucleus; arrowheads point to the cytoplasmic side of a nucleus; arrows point toward areas of increased fluorescence close to the poles of nuclei. (B) Fluorescence intensity profile obtained from the area delimited by the white rectangle in A. (C and D) TPLSM image sections of an FDB muscle transfected with pECFP-F and stained extracellularly with di-8-ANEPPS. ECFP-F and di-8-ANEPPS fluorescence images are shown in C and D, respectively. (E) Superimposition of image sections in C and D. (F) Normalized fluorescence intensity profiles measured from the area delimited by the rectangles in Fig. 2, C (cyan trace) and D (red trace). The vertical calibration bar is 20 µm and applies to all the images.

Action Potential–dependent QRET between EGFP-F and DPA
The fluorescence observed at the surface and TTS membranes in images of muscle fibers expressing EGFP-F can be detected as a steady level of intensity in quiescent fibers mounted for electrophysiological experiments in the fluorescence microscope. As shown in trace I of Fig. 3 A, this baseline fluorescence is insensitive to changes in membrane potential when the fiber is stimulated to elicit an AP (largest record in Fig. 3 B). The rest of the traces in Fig. 3 A illustrate the consequences that external perfusion with Tyrode containing 5 µM DPA has on the optical signals recorded from EGFP-F–transfected fibers. First, there is a progressive decrease in the baseline fluorescence reaching a steady state at 90% of the one previous to DPA exposure in 3 min (trace V); this decrement in fluorescence could be partially explained by the absorbance of excitation light by DPA staining the muscle membranes. In addition, it could reflect the steady-state QRET interaction between DPA and EGFP-F at the membranes. This latter possibility is reinforced by the detection of rapid negative changes in fluorescence (QRET transients; Fig. 3 A, traces II–V), which are elicited in response to AP stimulation (Fig. 3 B). QRET transients become progressively larger as the staining of the fiber membranes with DPA progresses in time. However, their amplitude reaches a maximum within 3 min (Fig. 3 A, trace V) after the initiation of the exposure to DPA. Both the magnitude of the basal fluorescence reduction and the maximal amplitude of the QRET signals were found to depend on the DPA concentration (unpublished data); furthermore, they were not reverted by DPA washout. The progressive increase in magnitude of the QRET signals after exposure of the fiber to DPA is better illustrated when these optical signals are expressed in F/F units and superimposed for comparison (Fig. 3 C, bottom). It can be observed that the peak F/F of the QRET signals increased from 1% when recorded 30 s after switching to DPA-containing solution, to 3% at the steady state. It should be noted that exposure to DPA is also accompanied with changes in the electrical properties of the muscle fiber. As illustrated in the top panel of Fig. 3 C, DPA induces an increase in the threshold for AP stimulation, a reduction in the amplitude of the AP, and a noticeable increase in AP duration. As will be shown later, these changes are expected and could be explained on the basis of the additional membrane capacitance contributed by DPA (Fernandez et al., 1983; Oberhauser and Fernandez, 1995). Keeping these considerations in mind, a simple explanation for the results reported in Fig. 3 (A and C) is presented in the scheme of Fig. 3 D. Due to its lipophilic properties and negative charge, DPA (yellow circles) partitions in a voltage-dependent manner across the surface and TTS membranes of the muscle fiber as it occurs in lipid bilayers and other excitable cells (Benz et al., 1976; Benz and Nonner, 1981; Fernandez et al., 1983). At negative potentials (i.e., the –90 mV resting membrane potential of the muscle fibers) the equilibrium distribution of dye molecules across the membrane favors their partitioning at the external leaflet, while membrane depolarizations increase the probability of their translocation toward the internal leaflet (Benz and Nonner, 1981). Furthermore, since DPA's absorption spectrum overlaps with the emission spectrum of EGFP-F (see Fig. 4), its translocation from the outer to the inner leaflet of surface and TTS membranes in response to the depolarization of the muscle fiber during the AP leads to an increased quenching of the EGFP-F fluorescence due to an increased QRET interaction between both molecules (Chanda et al., 2005). A corollary of this model, as suggested above and expanded later in the paper, is that there should be QRET between DPA and EGFP-F at the –90 mV resting potential of the muscle fibers since at this potential the probability of DPA being in the internal leaflet of the membranes is nonzero.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Steady-state and dynamic AP-dependent quenching of EGFP-F fluorescence by DPA. (A) Raw EGFP-F fluorescence before (trace I) and during DPA application (traces II–V). Records were acquired at 0, 1/2, 1, 2, and 3 min after DPA perfusion. Suprathreshold stimuli were delivered at t = 20 ms. Note the absence of QRET response in record I and the increasing amplitude of the AP-dependent fluorescence transients as perfusion time progresses. (B) APs that elicited the transients in A (the colors are matched). (C, bottom) F/F rendition (in an expanded time scale) of the QRET transients in panel A. (top) APs corresponding to QRET transients. (D) Schematic model of the voltage-dependent translocation of DPA and its interaction with EGFP-F. Different colors and length of the arrows were chosen to highlight the wavelength of excitation and emission, and the change in fluorescence intensity of EGFP. (E) Kinetic comparison of the largest QRET signal in A and C with its corresponding AP. The optical signal is shown inverted (–F/F).

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 4. QRET transient amplitude and DPA//EGFP-F and DPA//ECFP-F spectral overlap. (A) Spectral overlap between DPA absorption spectrum (red trace) and EGFP-F normalized emission spectrum (green trace). (B) Spectral overlap between DPA absorption spectrum (red trace) and ECFP-F normalized emission spectra (cyan trace). EGFP-F and ECFP-F emission spectra were determined in vivo and DPA absorption spectrum was measure in vitro. (C) Peak amplitude of EGFP-F (green hatched bar) and ECFP-F (cyan hatched bar) QRET transients evoked by AP stimulation. The averages values (in –F/F) were calculated from 6 and 12 fibers for EGFP-F and ECFP-F, respectively. The error bar is the SEM. The asterisk indicates statistical significance (P < 0.01). Signals were acquired using the filter combinations in Table I. (D) Average amplitude of the APs that elicited the QRET transients in C.

Spectral Overlap and QRET Signals
From FRET theoretical principles, a donor fluorophore, after being excited, transfers energy to an acceptor in a distance-dependent fashion according to the well-known formula (Stryer and Haugland, 1967; Clegg, 1995):

(1)

where E is the efficiency of energy transfer, R is the distance between the interacting molecules, and R_o is a constant whose value depends on their relative orientation and spectral properties. More precisely,

(2)

where J represents the spectral overlap of the donor emission with the acceptor absorbance, _D is the quantum yield of the donor, n is the refractive index of the medium, assumed to be 1.29 for membrane-bound probes (Selvin, 2002), and is a geometric factor accounting for the relative orientation of the transition dipoles of the donor and acceptor, which will be arbitrarily approximated to be 2/3 (Patterson et al., 2000; Selvin, 2002). The value of J was calculated using the formula (Selvin, 2002):

(3)

where _DPA() represents the molar absorption spectrum of DPA, calculated as the normalized absorption spectrum multiplied by the maximum molar extinction coefficient (20,000 M^–1cm^–1 in water), f_D() is the peak-normalized fluorescence emission spectrum of the donor, and is the wavelength. Fig. 4 A shows the spectral properties of the EGFP-F fluorescence superimposed with the normalized absorption spectrum of DPA, highlighting their relatively poor spectral overlap (hatched area). From these curves, a value of 2.8 x 10¹⁴ M^–1cm^–1nm⁴ was calculated for J^EGFP//DPA. By replacing this value in Eq. 2, and using the average measured value of _D = 0.6 for EGFP (Patterson et al., 2001), we calculate that for the EGFP-F//DPA pair, a value similar to the 3.7 nm reported previously (Chanda et al., 2005). Similar calculations, but using the absorption spectrum of DPA in methanol, which is slightly blue shifted with respect to that in water (unpublished data), yielded a 3% smaller value of . From Eqs. 2 and 3, it can be predicted that increasing the spectral overlap between donor and acceptor should result in a larger R₀ and thus to a more efficient energy transfer, potentially leading to larger QRET signals. Consequently, we decided to investigate whether ECFP-F significantly enhances the FRET interaction with DPA and, to this end, engineered a plasmid coding for this protein. As shown in Fig. 2 C, ECFP-F was identically expressed in the TTS membrane of muscle fibers as compared with EGFP-F. Fig. 4 B shows that in fact the in vivo spectrum of ECFP-F has a significantly broader overlap with DPA than EGFP-F (Fig. 4 A). The value of R₀ calculated from these data (ECFP-F//DPA pair), using the average measured value of _D = 0.62 for ECFP, was . This calculation resulted in 3% smaller values of when the methanol absorption spectrum of DPA was used. Interestingly, and in agreement with the expectations, pooled data show that on average the amplitude of AP-evoked QRET signals recorded from ECFP-F–transfected muscle fibers in the presence of DPA are significantly larger (approximately twofold) than those recorded at identical conditions from fibers expressing EGFP-F (Fig. 4 C). Panel D demonstrates that differences in QRET signals were not due to AP differences. Also, the difference in amplitude between ECFP-F//DPA and EGFP-F//DPA QRET transients were not dependent on the filter combination used to detect them since F/F values almost identical to those reported in Fig. 4 (within 3% error) were obtained in control experiments (not depicted) using six alternative filters sets with different emission bandwidths than those in Table I.

Interestingly, this value is larger than the nearly twofold ratio experimentally measured between F/F values for these QRET pairs (as shown in Fig. 4), suggesting that the average effective distance between acceptors and donors in the membrane are shorter than 1.7 x .

Voltage Dependence QRET between ECFP-F (EGFP-F) and DPA
The voltage dependence of QRET between ECFP-F and DPA was further studied under voltage-clamp conditions in fibers rendered passive by perfusion with V-clamp solution (see Materials and methods). Step voltage pulses were applied to fibers expressing ECFP-F or EGFP-F and stained with DPA; the evoked QRET transients were recorded simultaneously with membrane currents and potential. To assess the effects of DPA on the electrical properties of the muscle fiber, we compared the properties of capacitive currents before and after the addition of DPA to the bath. A typical example of current records acquired under control conditions in response to a family of depolarizing and hyperpolarizing pulses (10 ms duration) are shown in the top panels of Fig. 5 A. The bottom traces of Fig. 5 A show that the corresponding fluorescence records remain flat during the pulses, demonstrating that ECFP-F fluorescence, per se, is insensitive to depolarizations and hyperpolarizations to membrane potentials spanning the range from –150 to +50 mV. The addition of 5 µM DPA has visible effects on the current records and generates QRET signals in the optical records, as illustrated in Fig. 5 B. For depolarizing pulses, capacitive currents display a notorious enhancement of a slow decay component (top left panel) that was relatively small in records obtained under control conditions (Fig. 5 A); this is compatible with the presence of a drug-induced increase in the total membrane capacitance of the cell. For example, before DPA exposure, the relaxation kinetics of the capacitive transient elicited by a depolarizing pulse to +10 mV could be fitted to a double exponential function with I₁, ₁ and I₂, ₂ values of 0.97, 0.34 and 0.13, 2.49 (mA/cm², ms), respectively. These values changed to 1.28, 0.33 and 0.4, 2.46 (mA/cm², ms), respectively, after DPA, reflecting the enhancement of the slow component linked to an increase in the cell capacitance from 6.3 to 10 µF/cm² (60% increase). In contrast, the effects of DPA on capacitive transients elicited by hyperpolarizing pulses were negligible. A comparative analysis of QRET traces shows a marked asymmetry between the relatively large QRET transients elicited by depolarizing pulses (bottom left) and the small changes observed in the hyperpolarizing direction (bottom right). For example, the F/F amplitude of the QRET signal elicited by a depolarizing pulse to +10 mV signal was –4.3% whereas that one elicited by an identical pulse in the opposite direction (to –190 mV) was +0.7%. It should be noted that the ample nonlinear increase in the amplitude of the signals with the magnitude of depolarizing pulses contrasts with the crowding of QRET transients observed in response to hyperpolarizing pulses. It is also apparent that the asymmetry observed in the QRET records has an electrical equivalence since the total capacitive charge (measured by integration of the current) for a depolarizing 100-mV pulse (Fig. 5 B, top, blue trace) was 1.2 µC/cm², which contrasts with the 0.63 µC/cm² for the hyperpolarizing pulse of the same magnitude. Also, the rising phase of QRET records obtained with depolarizing pulses display faster kinetics ( < 1.3 ms) than the slow time constant of decay of the corresponding capacitive transients (₂ 2.4 ms). We will show later that this discrepancy is compatible with the predictions by a radial cable model of the TTS, where we believe the majority of the QRET signals originate.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Effects of DPA on the electrical and optical properties of a voltage-clamped skeletal muscle fiber expressing EGFP-F. The fiber was clamped at an HP = –90 mV and stimulated with depolarizing and hyperpolarizing voltage pulses of 40, 60, 80, and 100 mV. Data in A and B were obtained before and after DPA staining, respectively. (A) Top panels show capacitive currents in response to 10-ms depolarizing (left) or hyperpolarizing (right) pulses. Bottom panel shows, in an expanded ordinate scale, the corresponding ECFP-F fluorescence records. (B) Capacitive (top) currents and QRET transients (bottom) in response to 20-ms depolarizing (left) and hyperpolarizing (right) voltage-clamp pulses from the same fiber in A, after DPA staining. The rising phase of QRET transients elicited by depolarizing pulses could be adjusted to single exponential functions with = 0.4, 1.1, 1.1, and 1.2 ms for the black, red, green, and blue traces, respectively.

(4)

where Q_max (µC/cm²) is the total charge available to be moved (in this fiber 0.96 µC/cm²), V_h (mV) is the voltage at which half of the total charge is at each state across the barrier (+8 mV for this fiber), is the fraction of the electric field sensed by the mobile charge (0.84 for this fiber), HP (mV) is the holding potential, and F, R, and T have their usual meaning. The adequate fit of Eq. 4 to the data (Fig. 6 B, continuous curve) demonstrates that the DPA contribution to the current records broadly satisfies the expectations for the translocation of charges across the muscle membranes. An expected consequence of this charge translocation is that the capacitance (C_m) of the muscle fiber should be incremented by a voltage-dependent component. This is illustrated in Fig. 6 C. Before the addition of DPA (filled squares), the capacitance (obtained by dividing the current integral Q by the magnitude of the pulse) showed a relatively constant value typical of a linear capacitor, except for small voltage-dependent contributions given by the presence of voltage-dependent charge movements related to the excitation–contraction coupling and the gating of ion channels intrinsic to the muscle fibers (Schneider and Chandler, 1973; Simon and Beam, 1985a; Delbono et al., 1991; Kim and Vergara, 1998a). Overall, these charge movement contributions account for up to 25% deviation from a constant value of the recorded C_m as they are activated by both hyperpolarizing and depolarizing pulses from the HP (Adrian et al., 1976; Brum and Rios, 1987). In the case of the fiber in Fig. 6 C, the maximal contributions to C_m from these sources were estimated to be 20 and 10% in the depolarizing and hyperpolarizing directions, respectively. In contrast, the addition of DPA to the bath results in a significant increase in voltage-dependent capacitance (Fig. 6 C, open circles), which overwhelms the endogenous components. In the presence of DPA, we calculated the overall nonlinearity by fitting the data (represented by the open circles) to the equation

(5)

where C_V (µF/cm²) is the maximal voltage-dependent component of the capacitance, C₀ (µF/cm²) is the voltage-independent capacitance of the cell (measurable at very negative potentials at which the DPA contributions are minimized), V_h is the voltage at which the voltage-dependent capacitance is maximal, and the other parameters were as described above for the Boltzmann expression. Eq. 5, which follows the general definition of capacitance C = dQ/dV (Fernandez et al., 1983), is the explicit derivative of Eq. 4 with respect to the membrane potential plus the voltage-independent component C₀. Fitting Eq. 5 to the data in Fig. 6 C yielded values of 6.1 µF/cm², 5.4 µF/cm², 0.57, and 30 mV for C₀, C_V, , and V_h, respectively; the resulting curve is shown in the red solid line. Thus, the maximal voltage-dependent component of the capacitance relative to the capacitance at –190 mV grew from 9% before DPA staining to 5.4/6.1 = 89%, an 80% increase. It can be observed that DPA does contribute with a small percentage increase to the capacitance of the cell at negative potentials, in this fiber 0.3/5.8 = 5%, but this is negligible compared with that in the depolarizing direction.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Voltage dependence of DPA effects on steady-state electrical properties of muscle fibers. (A) Total charge mobilized in response to voltage pulses in a fiber expressing ECFP-F, measured before (control, squares) and after DPA staining (circles). The charge was calculated by integration at the on (open symbols) and off (filled symbols) of capacitive currents, normalized by the fiber surface area. The charge under control conditions was fitted to a linear regression (solid line, r = 0.9). (B) The difference between charge after DPA staining and the linear regression gives the extra charge contributed by DPA (on and off are open and filled circles, respectively). Data were fitted to a Boltzmann distribution using Eq. 4 (solid line). (C) Membrane capacitance before (filled squares) and after (open circles) DPA staining. The membrane capacitance at each membrane potential was calculated by dividing the charge (average of on and off) by the magnitude of the pulse. The capacitance after DPA staining was fitted to Eq. 5 (solid line).

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Voltage dependence of the FRET interaction between ECFP-F and DPA. (A) QRET transients (F/F) recorded in response to 20-ms voltage steps. The pulses were ±20, ±40, ±60, ±80, ±100, 120, 140, 160,180, and 200 mV. QRET transient amplitudes were measured as the average amplitude during a 2-ms interval, 2 ms after the onset of the pulses (indicated by the dotted rectangle). (B) Voltage dependence of the average QRET amplitude (filled circles). The error bar is the SD of the noise. Note that the ordinate is in –F/F units. The solid line represents the adjustment of the data to a Boltzmann distribution. (C) Extra charge contributed by DPA (on and off average) is shown in open circles. The error bars of the data points represent the SEM of on and off averages. The solid line corresponds to the adjustment of the data to a Boltzmann distribution. (D) Superimposition of average QRET transient and charge data, including adjusted curves. See text for details.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Dynamic FRET signals between DiBAC4(5) and EGFP-F. (A) EGFP-F emission spectrum (green trace) was measured in vivo and DiBAC4(5) absorption (orange trace) and emission (red trace) spectra were measured in vitro. They were plotted normalized for comparison. The orange dashed area represents the region of spectral overlap between the EGFP-F emission and DiBAC4(5) absorbance. (B) Fluorescence changes (top) elicited by two consecutive AP stimulation (bottom) recorded from a fiber expressing EGFP-F and stained with DiBAC4(5). Donor quenching (green trace) and acceptor-sensitized emission (red trace) are presented in the left and right panels, respectively; and were acquired consecutively by switching from the donor-EGFP to the sensitized-DiBAC4(5) filter sets (Table I). (C) The top and bottom panels show the EGFP-F and the DiBAC4(5) fluorescence changes elicited by 40-ms voltage pulses of ±20, ±60, ±100, 140, and 180 mV. The entire sequence of transients (applied at intervals of 1 min) was recorded first with the "donor-EGFP" cube and then with the "Sensitized-DiBAC" cube. Control records were obtained afterwards by alternating cubes for the same pulse amplitude (not depicted). (D) Voltage dependence of donor FRET (filled circles) and acceptor FRET (open circles). Signal amplitude was measured from averages during a 2-ms interval, measured 5 ms after the onset of the pulses. The error bar indicates the SD of the noise. Solid lines are Boltzmann fitting to the data. The characteristic parameters (F/Fmax, , and Vh) for the green/red sigmoidal curves were –0.09/0.6, 36.4/33.6 mV, and 15/0 mV, respectively. (E) Schematic model of the voltage-dependent translocation of DiBAC4(5) underlying its FRET interaction with EGFP-F. Different colors and length of the arrows highlight the wavelength of excitation of EGFP-F, and the change in fluorescence intensity of EGFP-F and DiBAC4(5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
In this paper we describe the first time use of FRET-based methods to measure membrane potential changes at the TTS system in mammalian skeletal muscle fibers. The method takes advantage of the ability of mammalian muscle to effectively express transgenic proteins in response to in vivo electroporation of DNA plasmids.

It had been previously demonstrated that muscle tissue can efficiently express recombinant soluble fluorescent proteins (e.g., EGFP and ECFP) following transfection of plasmids under the control of the CMV promoter (Dona et al., 2003; Umeda et al., 2004; DiFranco et al., 2006). Our current results demonstrate that muscle fibers can also efficiently express EGFP and ECFP constructs carrying a farnesylation signal; low magnification images show that up to 80–90% of the FDB muscle fibers were transfected (unpublished data). Most importantly, skeletal muscle fibers seem to provide the precise enzymatic environment to ensure that farnesylation occurs and that the EGFP-F and ECFP-F proteins are subsequently targeted to external membranes (surface and TTS membranes). These are critical features for an efficient hybrid FRET-based system since low amounts of fluorescent protein dissolved in the myoplasm (or associated with internal membrane compartments) ensure low background fluorescence and large signal-to-noise ratios in the optical data. Although EGFP-F and ECFP-F seem to be similarly targeted to both the surface and TTS membranes (Fig. 2), the FRET hybrid system reported here has the potential to be adapted for specific marker signals capable of differentially directing the expression of membrane proteins either to the TTS or the surface membrane. This would allow independent studying of these membrane compartments with standard light detection techniques, a goal previously approached by invasive detubulation procedures (Gage and Eisenberg, 1967; Heiny and Vergara, 1982).

QRET and FRET Signals Arise mostly from the TTS Membranes
It is well established that the electrical properties of muscle fibers are determined not only by the surface membrane but they reflect the important contributions from the TTS, which is a radial cable network connected to the surface membrane (Falk and Fatt, 1964; Adrian et al., 1969; Adrian and Peachey, 1973; Ashcroft et al., 1985; Kim and Vergara, 1998b). Thus, it seemed important to analyze whether radial cable model simulations of the muscle fiber (see Appendix) were able to predict the effects of DPA (and of DiBAC₄(5)) observed in the electrical records and if they provide a way to interpret the optical signals. As shown in the Appendix, we incorporated the kinetic equations of the two-state model for the voltage-dependent translocation of hydrophobic ions (Fernandez et al., 1983; Gonzalez and Tsien, 1995) to the surface and TTS membranes and computed the model predictions for capacitive currents and charge translocation by numerical integration of the partial differential questions using an implicit algorithm (Heiny et al., 1983; Ashcroft et al., 1985; Kim and Vergara, 1998b). The results of model simulations shown in Fig. 9 aimed specifically at predicting the data in Figs. 5 and 6, but were equally successful in fiber-to-fiber comparisons. Fig. 9 (A and D) shows data and model predictions, respectively, for the total charge translocation obtained in response to voltage-clamp pulses in a muscle fiber with and without DPA. It can be observed that model predictions (Fig. 9 D) are in good agreement with the experimental data obtained before and after the addition of DPA. Also, the model traces simulated in response to a 10-ms, +10-mV pulse are shown in the inset of Fig. 9 D to illustrate their similarity, both in amplitude and kinetics, to typical experimental records obtained with the same voltage. The deviations from the predicti