Monitoring voltage fluctuations of intracellular membranes

In eukaryotic cells, the endoplasmic reticulum (ER) is the largest continuous membrane-enclosed network which surrounds a single lumen. Using a new genetically encoded voltage indicator (GEVI), we applied the patch clamp technique to cultured HEK293 cells and neurons and found that there is a very fast electrical interaction between the plasma membrane and internal membrane(s). This discovery suggests a novel mechanism for interaction between the external membrane and internal membranes as well as mechanisms for interactions between the various internal membranes. The ER may transfer electrical signals between the plasma membrane and other internal organelles. The internal membrane optical signal is reversed in polarity but has a time course similar to that of the plasma membrane signal. The optical signal of the GEVI in the plasma membrane is consistent from trial to trial. However, the internal signal decreases in size with repeated trials suggesting that the electrical coupling is degrading and/or the resistance of the internal membrane is decaying.

The internal workings of the eukaryotic cell continue to astound. The ability of fluorescently tagged proteins to monitor the compartmentalization of the organelle interactome provides new insights into how the cell handles environmental conditions including stress 1 . A central player in the organelle interactome is the ER. The ER contacts every other organelle in the cell including the nucleus and plasma membrane [2][3][4][5] . This central role of the ER also makes it the stress center of the cell responding to unfolded protein accumulation (unfolded protein response -UPR) 6,7 as well as nutrient starvation and oxidative stress (integrated stress response -ISR) 8 .
Internal membranes involved in the compartmentalization of the cell may exhibit voltage changes 9 . In addition to the classical biochemical signaling cascades, electrical signals have also been postulated to send information from the cell's exterior to the nucleus 10 . Such a mechanism has remained speculative since the membrane potential of the ER cannot be directly measured by electrodes. Here we report the surreptitious trafficking of a GEVI that demonstrates the ability of electrical signals at the plasma membrane to affect the voltage of internal membranes.
Internal expression of plasma membrane proteins is usually a problem. For nearly a decade it was the bane of GEVIs 11 . GEVIs are fluorescent proteins which yield optical signals in response to changes in membrane potential. Using the voltage-sensing domain from a voltage-gated potassium channel, Siegel and Isacoff developed the archetype, FlaSh 12 . Using confocal microscopy and a voltage sensitive dye as a surface marker, Baker et al. 11 showed that FlaSh and two other first generation GEVIs were predominantly expressed intracellularly in mammalian cells probably due to protein misfolding. The protein was still fluorescent but did not traffic to the plasma membrane. As a result, no detectable voltage-dependent, optical signals were measured in mammalian cells with these sensors. This deficiency was overcome by using the voltage-sensing domain from the Ciona intestinalis voltage-sensing phosphatase 13 .
In this paper we introduce mutations to the loop regions of the Voltage Sensing Domain (VSD) of an ArcLight-type GEVI 14 resulting in a distinct intracellular optical signal that responds to changes in the plasma membrane potential. The internal signal for this GEVI, Aahn (Korean for inside), is always accompanied by an opposite polarity signal in the plasma membrane enabling simultaneous monitoring of internal membrane potentials in about 20% of the transfected cells. The other 80% of Aahn expressing cells do not exhibit enough internal expression to overcome the reverse polarity of the optical signal from the plasma membrane. The possibility that the other 80% of the cells do not experience a change in internal membrane potentials is real but relative low given that high intracellular fluorescence is an excellent predictor for observation of an internal signal. Regardless, this result indicates that an internal population of the probe is folded correctly, is functional, and demonstrates that alterations in the plasma membrane potential can affect the potential of internal membranes. This internal signal was also observed in neurons suggesting that this phenomenon may be present in many if not all cell types.

Results
ArcLight mutations: External and internal optical signals from HEK293 cells and hippocampal neurons. With the aim of adjusting the voltage-sensitivity of GEVIs, over 180 unique voltage sensing domains from voltage-gated sodium channels (Na v ) were aligned to identify highly conserved amino acids in the cytoplasmic and extracellular loops connecting the transmembrane segments.  15 , yielded probes which have an internal signal distinct from the external, plasma membrane signal. In Fig. 1A, the voltage sensing domain is shown consisting of the four transmembrane segments, S1-S4. The yellow regions indicate the location of these mutations that generated an internal signal. These data suggest that some mutations in the intra/extracellular loop regions of the VSD behave like a new motif for expression in intracellular membranes. However, this novel mechanism does not seem to inhibit the expression of the protein in the plasma membrane. Introduction of two mutations near the external transmembrane/loop junction of S1 (P140S and G141D) into the GEVI, ArcLight, resulted in a new probe, Aahn, capable of optically monitoring both plasma membrane and internal membrane potentials. Figure 1 illustrates the opposite signed fluorescence signals in response to manipulating the plasma membrane potential. The plasma membrane fluorescence gets dimmer (blue trace) during depolarization steps while the internal signal (red trace) gets brighter when expressed in HEK cells. Similarly, Fig. 1 also illustrates a similar opposite signed fluorescence response in a cultured neuron. Since the internal signal is reversed compared to the plasma membrane signal, we suppose either that when the plasma membrane is depolarized, an internal membrane is hyperpolarized, or that the GEVI in the internal membrane is reversed in direction.
HEK293 cells are connected to each other electrically via gap junctions. To avoid physical contact between the patch electrode and internal membranes, a neighboring cell was patched and voltage clamped (indirect patch clamping, e.g., Fig. 1B). Patching and recording from the same cell is designated as direct patch clamping. Direct patch clamping was used for neurons. We carried out 4 or 6 repetitions with four trials in each repetition. Unless indicated, the results come from the average of the four trials in the first of the repetitions.
Frame subtraction images identify the location of a distinct, internal, voltage signal. Subtraction of fluorescence intensities of frames prior to a voltage step from the intensities of the frames during the voltage step (black bars in Fig. 1E,H) results in an image identifying the location of the optical signals. We presume that at each pixel the signal is the summation of the internal signal and the external signal. Near the edge of the cell, the external signal should predominate. In the center of the cell, an internal signal will be detected if it is larger than the external membrane signal at that pixel. In Fig. 1D,G, the white area indicates regions where the internal membrane signal predominates while the black areas represent areas where the external signal predominates. We name the reversed signals as "internal signals". The internal signal is likely obscured by the external signal in other locations. The fluorescent intensity decrease at the edge of the cell during depolarizations (dark areas in Fig. 1D,G) is typical for ArcLight. In this and subsequent figures the blue trace indicates the plasma membrane signal and the red trace indicates the internal membrane(s) signal. The results illustrated in Figs 2-5, were carried out in HEK293 cells.
Overlap of the internal signal with the nucleus. Additional results from the HEK293 cell illustrated in Fig. 1 are shown in Fig. 2. In the experiment illustrated in Fig. 2A-E, HEK293 cells were co-transfected with a red fluorescent marker targeted to the nucleus (nls-mCherry) 16 together with Aahn. The neighbor cell was voltage clamped and subjected to negative and positive voltage steps. The Resting Light Intensity (RLI) of this cell shows high intracellular expression ( Fig. 2A). There is no obvious overlap between the internal signal area and the nucleus marker (Fig. 2D). Either a nuclear envelope optical signal is obscured by the plasma membrane signal, or Aahn is not present in the nuclear membranes, or the nuclear membrane does not experience a voltage change. One frame of the 3 dimensional visualization of the frame subtraction during the 100 mV depolarization pulse is shown in Fig. 2E. Overlap with the ER. Co-localization was observed in HEK293 cells expressing both Aahn and an ER marker, Sec. 61, fused to a red fluorescent protein, mCherry 17 (Fig. 3). As shown in Fig. 3A, a neighbor cell was voltage clamped and stepped with negative and positive voltages. Figure 3B shows high internal fluorescence with some overlap with Sec. 61-beta fused to the fluorescent protein, mCherry, as an ER marker (Fig. 3C). Figure 3D shows evidence of the Aahn internal membrane response (white) and the external membrane response (black) to the100 mV depolarization pulse. Figure 3E shows one frame of the three dimensional visualization of the frame subtraction during the 100 mV step. The overlap of the internal signal with the ER is not perfect, likely due to the fact that the white region only indicates the locations where the internal signal is larger than the external signal. None-the-less, the result shows that the ER is a possible source of the internal signal. Additional examples are shown in Supplemental Fig. S1.
Confocal imaging. HEK293 cells co-expressing Aahn with the ER marker (mCherry-Sec. 61-beta) were imaged with a confocal fluorescence microscope to obtain higher resolution images of the overlap. The ER marker overlaps with the internal Aahn expression when Aahn's internal expression is high (Supplemental Fig. S2). These results suggests that the ER is a possible source of the Aahn internal signal. Cells with limited Aahn internal expression were usually not selected for patch clamp-fluorometry recordings.
Comparison of external and internal time courses. Using single and double exponential fitting of the Aahn response to 100 mV steps we estimated the external and internal signal time constants for five cells avoiding pixels near the border between the reversed internal and external signals (Supplemental Table S1). The averaged response from three pixels which had the largest signal was used for both internal and external signals. The average difference in time constants between external and internal signals was small and not significantly different (p = 0.78) (Supplemental Table S1). However, small differences in the time courses of the internal and external signals sometimes resulted in unusual signals at the border between the external and internal regions (Supplemental Fig. S3). The unusual signals are most noticeable during larger depolarizations. Unusual signals at the boundary between internal and external signals were seen in 6 out of 9 HEK293 cells. Supplemental Fig. S4 shows the relative change in fluorescence of the internal and plasma membrane signals as a function of membrane potential. The external and internal signals have very similar voltage dependences.
Effect of repeated trials. The internal signal decreased in size during repeated patch clamp measurements while the external signal remained unchanged (Fig. 4). We envision two causes for the decrease in the internal signal: (1) The internal membrane(s) resistance decreases during patch clamp measurements and/or (2) The connection between the internal membrane(s) and plasma membrane is dissipating. The rate of internal signal decrease for indirect patch clamping is smaller than for direct patch clamping (Fig. 5A,B). Using only −50 mv and +50 mv voltage pulses (Fig. 5C) or a more depolarized holding potential (Fig. 5D) did not change the rate of decrease of the internal signal. The internal signal area also shrinks during repeated patch clamp measurements. This internal signal area change is likely due the internal signal size reduction. Since the external signal size remains unchanged and the internal signal size decreases, the area with reversed signal will decrease with repeated measurements. It is unlikely that the decrease observed over several repetitions is due to the accumulation of excess charge in the internal membranes since alternating the hyperpolarizing and depolarizing pulses showed a similar result (Supplemental Fig. S5).

Discussion
We have optically monitored voltage changes in internal membrane(s) and the plasma membrane simultaneously. Using a new genetically encoded voltage indicator, Aahn, we applied the patch clamp technique to HEK293 cells and cultured neurons and found that there is an electrical interaction between the plasma membrane and the internal membrane(s) of both cell types. The ER is both a continuous membrane bound organelle and has components that are closely opposed to and interacting with the external membrane. Thus, it is very likely that the ER contributes to Aahn's internal signal. The Golgi apparatus is also widespread in the cytoplasm but, in contrast, is not thought to exist as a continuous membrane bound organelle and is not thought to have close oppositions to the external membrane (M. Terasaki, personal communication). Any Golgi contribution to Aahn's internal signal is likely to be secondary. The voltage changes in the internal membrane(s) are highly synchronized with the voltage changes in the plasma membrane (Figs 1-4). In a previous report from our lab, we have shown that pH induced optical signals are very slow suggesting that Aahn's internal signal is voltage dependent 18 . In eukaryotic cells, ER-PM junctions can be as close as 10 nm 19 and play a role in the movement of ions and lipids 20 . The voltage-gated potassium channel, Kv2.1, has been shown to direct the architecture of the ER to the PM in both HEK cells and neurons 21 . We speculate that these sites of close apposition could couple voltage alterations of the PM to the ER. One mechanism could be via direct fusion of the ER to the plasma membrane. During calcium depletion of the ER, an ER resident protein, STIM1, is activated and binds to a protein in the plasma membrane ORAI 22 . Reports in Jurkat T cells 23 and in Xenopus larvae hair cells 24 demonstrated that staining the external leaflet of the plasma membrane with the membrane-impermeable styryl dye, FM1-43 resulted in the staining of the lumen of peripheral ER suggesting the possibility for ER-PM fusion. In a second mechanism the close proximity of the ER and PM at these junctions In addition to the signaling network represented by the second messenger cascade, here we have shown that an internal membrane, potentially the ER, can act like a "cytoplasmic nervous system" which conducts electrical signals for intracellular communication 9 . One advantage of such a direct electrical communication is the high speed of this process.
The following reports might possibly be explained by electrical connections between external and internal membranes. Friedman et al. 27 found that mitochondrial division occurs at positions where ER tubules contact mitochondria in both yeast and mammalian cells. Caldieri et al. 28 showed that epidermal growth factor receptor endocytosis relies on ER-PM contact sites and local Ca (2+) signaling. ER-PM contact sites have also been implicated in regulating recycling of potassium channels 29 .
The expression pattern of Aahn is also interesting in that accumulation of intracellular expression does not seem to inhibit trafficking to the plasma membrane. Aahn clearly expresses well at the plasma membrane. The voltage-dependent optical signal is quite robust (Figs 1E,H,G,D, 3F and 4D). Indeed, the plasma membrane expression is a potential problem since the reverse polarity of the optical signal at the plasma membrane likely masks the internal signal accounting for our ability to only detect an internal signal in 20% of transfected cells.  Using GEVIs to monitor internal membrane potential changes may help to understand the regulation of the organelle interactome 30 and improve our understanding of how the cell responds to its environment and stress. We expect that this approach can be used to enhance our view of the role of the ER and other internal organelles in cell physiology both in health and disease.

Materials and Methods
Plasmid DNA designs and construction. nls-mCherry was a gift from Jinhyun Kim (Addgene plasmid #34911) 16 . DNA sequences for all of the constructs were confirmed by DNA sequence analysis using the dye-termination method (Cosmogenetech  Fig. 5D where the holding potential was −35 mv. Wide-field imaging. Whole-cell patch clamped cells were imaged with an OlympusIX71 microscope with a 60 × 1.35 numerical aperture oil-immersion lens (Olympus). A 75 W xenon arc lamp (Cairn Research) was used as the excitation light source. The excitation filter for the green fluorescence protein (Aahn) was 472/30, the emission filter was 496/LP and the dichroic was 495 (Semrock, NY). The excitation filter for the red fluorescence protein (mCherry-NLS) was 562/40, the emission filter was 641/75 and the dichroic was 593 (Semrock, NY). The fluorescence image was demagnified by an Optem zoom system, A45699 (Qioptiq LINOS) and projected onto the 80 × 80 pixel chip of a NeuroCCD-SM camera controlled by NeuroPlex software (RedShirtImaging, GA). The images were recorded at a frame rate of 500 fps. The bright field images were demagnified by an Optem zoom system, A45699 (QioptiqLINOS) and captured by a (Hitachi, Tokyo) CCD camera (KP-D20BU). The frame grabber (PCI-RTV24) and its corresponding software (ADLINK ViewCreatorPro) was used to control the Hitachi camera and record the images.
Confocal Imaging. Using a Plan-Apo 60×/1.40 Oil DIC N2 objective and a Nikon Eclipse Ti (Nikon, Japan) confocal laser scanning microscope, confocal images were obtained. A 488 nm wavelength laser was used for Aahn excitation. The emission filter for the Aahn was 500-550 nm. A 561 nm wavelength laser was used for ER marker (mCherry-Sec. 61-beta) excitation. The emission filter for these red florescent proteins was 570-620 nm. NIS-Elements microscope imaging software was used for image acquisition and processing.
Optical signal analysis. We used NeuroPlex software (RedShirtImaging) to calculate the ∆F F % / , the dark image was subtracted from all frames, then the average of a region of interest in each frame F ( ) is subtracted from the average of the region taken from ten frames prior to the event of interest F ( ) 0 and then this value was divided by