Department of Physiology, University of Arizona, Tucson, Arizona
Submitted 19 April 2005 ; accepted in final form 4 August 2005
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ABSTRACT |
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connexin 43; connexin 37; diffusion rate constant
To understand why the mammalian genome includes 20 or more connexin genes, the functional differences between the gap junctions that they form must be appreciated. Since their discovery, much has been learned about gap junctions in a broad sense, but their functional and regulatory differences are still largely unexplored. Regardless of connexin composition, all gap junctions mediate electrical signaling, and most, as implied by the intercellular diffusion of fluorescent dyes, mediate chemical signaling. In studies in which they have been examined quantitatively, the small ion charge selectivity of gap junction channels has been found to be weak (<10-fold) or absent. For example, Cx43 channels in their full open state display virtually no preference for small anions vs. cations (9, 37); in contrast, Cx40 channels display a fivefold preference for K+ over Cl (1). Despite this weak preference of Cx40 channels for cations, the presence of permeant anions reduces the measured single-channel conductance (j), suggesting that they impede the flow of cations when present. Whether the cation/anion preference of gap junction channels is more extreme for molecules near the size limit of the channels pore has not been explored quantitatively, but were this the case, it could explain the sometimes opposite effects on dyes vs. electrical coupling that kinase activation or altered connexin expression can have (2023). Decreased dye coupling in the face of unchanging or increasing electrical coupling has been interpreted by various groups as evidence for differential regulation of electrical vs. metabolic coupling. Instead, such observations may reflect regulated (graded) selectivity. The available data support the contention that regulation of junctional selectivity is vital for the appropriate response of cells to interventions of various types. Tissue injury (e.g., as a consequence of ischemia) and repair are settings in which it could be advantageous to maintain electrical coupling (i.e., diffusion of small ions) and to some degree metabolic coupling (i.e., diffusion of nutrients) while restricting the passage of bigger or uniquely shaped potentially toxic substances. Importantly, such graded changes of selectivity would likely be missed if junctional coupling were assessed with either nonquantitative dye-coupling methods or electrical coupling techniques in which KCl or CsCl comprised the major current-carrying ions (use of larger current-carrying ions would increase the likelihood of detecting differences; Ref. 20). The strategy presented herein overcomes many of the limitations of previously used techniques to quantify the selectivity of gap junctions as a function of their connexin composition or in response to interventions that might regulate junctional selectivity (8, 33).
Our strategy for quantifying junctional selectivity improves previous methods (35) by determining the rate constant for transjunctional dye diffusion (k2-DYE) and the junctions conductance (gj), such that dye selectivity, defined as (k2-DYE/gj), can be calculated and correlated with the properties of the contributing channels. Through judicious selection of dyes (different charge and/or size), it should be possible to define (quantitatively) connexin-specific differences in selectivity. Possible alterations of selectivity that might occur after activation or inhibition of signaling cascades can be evaluated, and the requirement for specific sites in the connexin protein can be established by site-directed mutagenesis. Thus the multifaceted approach described herein can be expected to yield new insights into the regulation of cell-cell communication through acute and long-term alterations of selectivity.
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METHODS |
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Rat insulinoma (Rin) cells were stably transfected with rat Cx43 [rCx43; Rin43 expression driven by cytomegalovirus (CMV) promoter; Ref. 36] or transfected with pTET-ON, stable clones isolated by dilution cloning, and one such clone was subsequently transfected with pTreHygro-mCx37 (iRin37). Doxycycline treatment induced murine Cx37 (mCx37) expression in a dose-dependent manner using a 24 µg/ml for 2448 h, resulting in maximal expression (data not shown). Rin cell lines were grown in RPMI 1640 (R1383; Sigma) supplemented with 10% FBS (Gemini BioProducts), 300 µg/ml penicillin G, 500 µg/ml streptomycin, 300 µg/ml G418 (and 100 µg/ml hygromycin for iRin37), and maintained at 37°C in a 5% CO2 humidified incubator. Normal rat kidney epithelial (NRKE) cells, which naturally express Cx43, were grown in DMEM (D-1152; Sigma) supplemented with 4.5 mg/ml glucose, 10% FBS (Gemini BioProducts), 300 µg/ml penicillin G, 500 µg/ml streptomycin, and 300 µg/ml G418, and maintained at 37°C in a 5% CO2 humidified incubator. Rin or NRKE cells from confluent plates were lifted by trypsin treatment, transferred to glass coverslips, and allowed to adhere. Coverslips with adherent cells were mounted in a custom-made chamber and placed on the stage of an upright microscope (BX50WI; Olympus) equipped for differential interference contrast (DIC) and fluorescence observation. Cell pairs in which both cells were of similar size and shape were selected for experiments.
Fluorescence vs. Dye Concentration
In the experiments described herein, two dyes were used: gap junction-permeant NBD-M-TMA+ {N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]ethanaminium, charge 1+; molecular weight, 280 Da; ex = 343,458 nm;
em = 530 nm} (2)1 and gap junction-impermeant rhodamine-labeled dextran (3,000-Da dextran tetramethyl rhodamine,
ex = 555 nm,
em = 580 nm; Molecular Probes). Fluorescence was visualized using the Olympus BX50WI microscope outfitted with a fluorescein filter set (U-MNB; Olympus) for NBD-M-TMA+ detection and a rhodamine filter set (U-MWG; Olympus) for rhodamine dextran detection. In both cases, a SenSys charge-coupled device (CCD) camera captured digital images that we analyzed using V++ software (Photometrics). To define the relationship between NBD-M-TMA+ concentration and detected fluorescence, we used a hemocytometer to trap a fixed (10 µl) volume of the dye at various concentrations and captured images using this system. The average fluorescence intensity (U-MNB filter set) detected in each column of the CCD array for NBD-M-TMA+ concentrations between 0 and 0.250.5 mM and exposure times between 25 and 500 ms was obtained. As shown in Fig. 1A, fluorescence intensity increased linearly with increasing concentration for each exposure time. In addition, the fluorescence detected for a specific concentration increased linearly with increasing exposure time (Fig. 1B). Results comparable to those displayed in Fig. 1, A and B, for fewer dye concentrations and sampling times were observed using a slide with a coverslip instead of the hemocytometer as the sample chamber (data not shown). On the basis of preliminary experiments with cell pairs, we found that a sampling time of 250 ms and a pipette dye concentration of 0.25 mM were optimal, yielding fluorescence levels far above the water control but below the cameras saturation level. At this concentration and exposure time, cells injected only with NBD-M-TMA+ could sometimes be detected with the rhodamine filter set. The magnitude of the NBD-M-TMA+ signal detected by the rhodamine filter set was
8% of that detected using the fluorescein filter set (Fig. 1C).
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Voltage Control with Discontinuous Single-Electrode Voltage-Clamp Amplifiers
Discontinuous single-electrode voltage-clamp (DSEVC) amplifiers alternate between current injection and voltage-recording modes at switching frequencies of 170 kHz (set by the user). At the switching frequency of 25 kHz typically used in our experiments, the amplifier injected current for 5 µs and sampled the membrane potential 25,000 times/s for 35 µs. For each of these cycles, the amplifier adjusted the injected current such that the measured membrane potential equaled the user-specified holding potential. These amplifiers have already proved useful for recording junctional current (Ij) in well-coupled cell pairs (27). In Fig. 2, the voltage control achieved using these amplifiers is displayed. In Fig. 2A, we show that the head-stage voltage and amplifier output were comparable. Depolarizing pulses were alternately applied to either cell of a well-coupled pair, while the partner cell was kept at 0 mV and the electrode potential (Vel, taken from the back of each amplifier) was compared with the internally filtered voltage output (Vout). For visual purposes, the dot size of the Vel traces was enhanced. Only traces from one cell are shown; stability at the 0-mV holding potential while the pulse was applied to the opposite cell indicated the establishment of a well-controlled voltage gradient across the junction. Note that the Vel and Vout deflections were equivalent in amplitude (the difference in their appearance reflects the absence in the latter of frequencies >13 kHz, which was the filter setting for this recording). Figure 2B shows, for the same cell pair, that the holding potential was achieved early in a 10-ms voltage step. Depolarizing voltage pulses were simultaneously applied to each cell; Vout for cell 1 and the corresponding unfiltered Vel are displayed. Clearly, voltage stabilization and complete voltage control were achieved in <2 ms. These results demonstrate that each cells voltage, and therefore Vj, was superbly controlled with these amplifiers. The actual switching frequency used in any given experiment (1535 kHz) was chosen to minimize electrical noise. The amplifiers were designed to work with high-resistance electrodes (32); consequently, the low-resistance electrodes used in our experiments needed minimal capacitive compensation and allowed high levels of current delivery. It is worth noting that these amplifiers minimize the chances of underestimating (as a result of high electrode resistance) gj in well-coupled pairs as occurs with traditional continuous single-electrode voltage-clamp (CSEVC) amplifiers. When instability of voltage control occurred, the presumed junction turned out to be a cytoplasmic bridge (see Fig. 4 and associated text for criteria used to discern cytoplasmic bridges). Data from such experiments were discarded.
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Determining junctional dye permeability.
Patch-type microelectrodes were 512 M when filled with recording solution (in mM: 124 KCl, 14 CsCl, 9 HEPES, 9 EGTA, 0.5 CaCl2, 5 glucose, 9 tetraethylammonium-Cl, 3 MgCl2, and 5 Na2ATP, adjusted to
320 mosM with H2O and pH 7.2 with KOH) (7). The electrode for the donor cell also contained 0.25 mM NBD-M-TMA+ and 1 mg/ml rhodamine-labeled dextran. Because NBD-M-TMA+ fluorescence was linearly related to its concentration, we selected cell pairs that were of similar size and shape (and therefore of similar volume), such that cytoplasmic dye concentration and cell fluorescence could be equated (which facilitated the modeling of the intercellular k2; see below). A G
seal was obtained with the donor cells electrode, and the cytoplasm was accessed using gentle suction or an electrical buzz. Access was immediately obvious on the basis of rapid entry of NBD-M-TMA+ (fluorescein filter set) into the donor cell. Intercellular diffusion of the dye was documented by acquiring fluorescent digital images of the cells every 1015 s for the first minute and every 13 min thereafter for up to 20 min. The rhodamine dextran image was typically acquired after electrical measurements (see below) were obtained, because shifting to the rhodamine filter set often compromised the G
seal on the donor cell. However, when intercellular NBD-M-TMA+ diffusion was fast, often indicative of a cytoplasmic bridge, the rhodamine dextran image was acquired before electrical measurements were attempted. If dextran was detected in the recipient cell (at a level significantly greater than the 8% level expected from the presence of high levels of NBD-M-TMA+), the experiment was discarded. If dextran was not detected in the recipient cell, gj and
j data were acquired (see below).
Determining gj and j.
Two current-voltage switching-clamp amplifiers (SEC-05LX; NPI Electronic) (27, 32) were used to determine gj and
j. After documenting intercellular dye diffusion, a G
seal with the recipient cell was made with a second electrode filled only with recording solution. For both donor and recipient cells, seal formation was monitored in current-clamp mode with
200-pA current pulses of 60-ms duration delivered at a frequency of 35 Hz. Comparable pulses delivered at a reduced frequency, typically 0.2 or 3 Hz, were applied to the donor cell throughout the dye transfer period to monitor seal quality. After gaining access to the interior of the recipient cell, both amplifiers were switched to voltage-clamp mode and macroscopic Ij was documented during 10-mV depolarizing pulses applied alternately to each cell. Halothane (5.5 mM) was superfused or dripped on the cell pair to induce electrical uncoupling, and channel activity was recorded with Vj = 4050 mV for Cx43- and Vj = 25 mV for Cx37-composed junctions (Vj selected to be less than corresponding V0, where V0 is the voltage at which the voltage-sensitive component of gj is reduced by 50%). Pulse protocols were driven using either an A-65 timer (Winston Electronics) and two S-100 direct current (WECO) power sources or Digidata 1322A and Clampex software (pClamp8; Axon Instruments; and an Intel Pentium personal computer; Dell). Signals were digitized (Neurocorder DR-484; Neurodata Instruments) for storage on videotape and simultaneously filtered directly (LPF202A, 100 Hz; Warner Instrument) and stored on the computers hard drive for subsequent analysis.
Data Analysis
Junctional dye permeability. Fluorescent images were stacked in chronological order and rotated to align the cell-to-cell contact margin with an imaginary y-axis. A rectangular region of the CCD array that encompassed both cells and some noncellular area (background) was electronically defined and superimposed on the image, which is shown in Fig. 3A. The V++ software returned the total or mean fluorescence detected in each column of the array within the rectangle (plotted in Fig. 3A for each acquisition time). Because fluorescence intensity across the cells area varied as a result of variation in cell thickness and therefore the sampled volume, we selected a column near the center of each cell and defined the analyzed rectangle to include 515 columns on either side; the number of columns was chosen according to cell size and typically encompassed >50% of each cells volume. The mean fluorescence for these equivalent areas (i.e., volumes) of the donor and recipient cells was determined and plotted as a function of time. The time course of fluorescence increase (recipient-to-donor ratio) was nearly identical, regardless of whether total cell fluorescence or fluorescence from limited volumes of each cell was plotted; however, the use of equivalent limited volumes minimized the influence of the electrode tip fluorescence.
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Detection of cytoplasmic bridges. Gap junctions are not permeated by 3,000-Da rhodamine dextran, are reversibly uncoupled by halothane, and exhibit single-channel activity, none of which is true for cytoplasmic bridges. Consequently, these criteria can be used to distinguish cytoplasmic bridges from true gap junctions. The transfer of NBD-M-TMA+ through cytoplasmic bridges was typically fast and abundant (Fig. 4A), resulting in donor and recipient cell fluorescence being nearly equal at all sampled time points. When such rapid transfer was observed, a rhodamine dextran image was typically acquired before electrical measurements were pursued. If the rhodamine dextran image revealed similar levels of fluorescence in the donor and recipient cells, the experiment was terminated because such junctions were always cytoplasmic bridges on the basis of the criteria stated above.
Even cell pairs that had a flat, extended appearance and therefore seemed unlikely to be joined by a cytoplasmic bridge were nevertheless sometimes separated by a bridge (Fig. 4B). In this case, the NBD-M-TMA+ intercellular diffusion rate, 0.465 min1, was in a range (see F/Fmax plot) that was fairly common for true gap junctions; consequently, gj was measured and halothane-induced uncoupling was attempted. This pair could not be uncoupled with repeated exposure to halothane, and intercellular conductance was unsteady, spontaneously decreasing from 3 to 0.5 nS (not shown). The rhodamine-labeled dextran image revealed nearly equal levels of rhodamine fluorescence in donor and recipient cells, even after extended whole cell clamping of the recipient cell, confirming that a cytoplasmic bridge rather than a gap junction separated the cells.
Occasionally, a low level of rhodamine fluorescence was detected in a recipient cell, despite evidence (reversible, halothane-induced uncoupling and/or gap junction channel activity) that a gap junction rather than a bridge joined the cells. This occurred only in highly coupled pairs and most likely arose from a combination of two artifacts. First, the 3,000-Da rhodamine-labeled dextran preparation is not homogeneous (molecular weight dispersion in the 1,500- to 3,000-Da range) and may contain molecules of <1,500 Da, including free dye (15). Because Cx43 channels are permeated by rhodamine (10, 17), this free dye may be detectable in the recipient cell, especially when coupling levels are high and experimental durations are long. Second,
8% of the fluorescence emitted by NBD-M-TMA+ occurred at wavelengths detected by our CCD camera through the rhodamine filter set (Fig. 1C). Correction of the rhodamine signal for this NBD-M-TMA+ fluorescence always reduced the apparent rhodamine signal to near background levels when a gap junction rather than a bridge joined the cells.
gj and j.
Macroscopic and unitary gj values were calculated using Ohms Law (gj = Ij/Vj or
j = Ij/Vj), where Ij was the current delivered by the amplifier controlling the nonpulsed cell during application of Vj, the transjunctional voltage gradient. Measurements were obtained from the pClamp recordings (see, e.g., Fig. 6, A and B) and analyzed using Excel, SigmaPlot, and Origin software as is customary (7).
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RESULTS |
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After accessing the donor cells interior, fluorescence in the donor cell approached an asymptotic maximum determined on the basis of the dyes concentration in the pipette. Figure 5 shows NBD-M-TMA+ intercellular diffusion data obtained from two pairs of Rin43 cells. In both pairs, fluorescence was readily detected in the recipient cell at the 1-min time point and increased with little delay as the fluorescence in the donor cell increased. For the pair shown in Fig. 5A, the recipient cells fluorescence data (plotted as the fluorescence at each time point relative to the maximum observed fluorescence, F/Fmax) were well fit, with a k2 of 0.52 min1. That this value represented the best fit was evident from the plot of RMS vs. k2 at the bottom of Fig. 5A; clearly, a k2 value that differed by only 0.01 min1 in either direction noticeably increased the RMS, indicative of a less satisfactory fit. Filling of the donor cell in Fig. 5B was initially slow because of incomplete rupture or partial sealing over of the electrode. At minute 5, the donor cells electrode was buzzed a second time, which resulted in a faster filling rate. Fluorescence in the recipient cell tracked that of the donor with little delay as shown in the F/Fmax plot. Despite this change in donor cell-filling rate (k1), a single rate constant for intercellular diffusion (k2) described the filling of the recipient cell. The best fit, evident from the RMS plot, was obtained with a k2 of 0.75 min1. These data demonstrate the efficacy of our model in detecting k2 in a manner independent of potential changes in k1. Comparable data were collected from 26 pairs of Rin43 cells in which gj 1 nS and rate constants ranged from 0.052 to 2.9 min1.
gj and j
Once intercellular dye diffusion had been documented with enough images to obtain a valid k2 (typically 710 images acquired in 10 min), an electrode was placed on the recipient cell and gj was determined. Although gj could change spontaneously in this period of time, available data from this and many other laboratories indicate that the conductance of Cx43 (and many other connexin types) composed junctions is stable over recording periods of this duration in whole cell clamp mode. That intercellular dye diffusion was consistently described by a single rate constant lends further support to the stability of junctional function during the recording period. Junctional conductance was measured at the conclusion of the dye diffusion period immediately after accessing the interior of the recipient cell, during exposure to halothane, and during halothane washout as shown in Fig. 6 for the cell pairs in Fig. 5. Figure 6A shows (for the cell pair in Fig. 5A) the macroscopic currents for recipient (top trace) and donor cells (bottom trace) before halothane exposure (left), as halothane was applied (center; spikes in trace represent mechanical artifact of perfusion), and after (right) complete uncoupling (Ij is indicated by downward deflections). No channels were observed during halothane washout before loss of seal on the donor cell in this experiment. Figure 6B shows similar data for the cell pair shown in Fig. 5B. In this case, halothane superfusion was initiated during the first set of pulses shown (electrical spikes indicative of perfusion-induced mechanical artifact); several seconds later, the cells were uncoupled (second set of pulses). The third set of pulses was obtained after partial recovery from halothane-induced uncoupling (
5 min later, Vj increased to 40 mV). The channel activity observed during exposure to halothane in this and two other cell pairs is shown in Fig. 5C. Single-channel recordings and associated all-points histograms obtained from the cell pairs shown in Fig. 5B (top trace) and from two other cell pairs revealed a diversity of channel amplitudes in Rin43 cells. A diversity of channel amplitudes was also observed for NRKE cells that endogenously express Cx43 (data not shown).
Changes in k2 Are Readily Detected
For 26 Rin43 cell pairs, our model calculated a single k2 for intercellular diffusion of dye. The quality of fit obtained in our experiments indicates that k2 does not change significantly during the course of the typical experiment. To determine whether our methodology was capable of discerning a change in k2 if intentionally induced, we applied halothane while measuring intercellular dye diffusion. As shown previously (6) (Fig. 6), halothane causes a reduction in gj, most likely through a change in channel open probability (6). Figure 7 shows the F/Fmax as a function of time plot for an experiment, in which halothane was applied 5.5 min into the recording period. The plot shows that fluorescence in the donor cell continued to increase after halothane application, whereas fluorescence in the recipient cell stabilized until halothane washout was initiated at 7 min. The data from the first 5 min were best fit with a rate constant of 0.85 min1. On the basis of the donor cells fluorescence, this rate constant was used to predict what the recipient cells fluorescence should have been if k2 were unaffected by halothane (closed circles and solid line). Clearly, the recipient cell data obtained after application of halothane were not well fit by this single rate constant. The second rate constant reported in this figure, k2 of 0.285 min1, represents the best fit of the recipient cell fluorescence data obtained after partial washout of the halothane. In four such experiments, full recovery of the rate constant was not observed. Nevertheless, this experiment demonstrates that our approach to determining the permeability of a gap junction is sensitive to rate constant changes that might occur during the course of the experiment and, importantly, indicates that the stability of the rate constant is indicative of junctional stability.
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Cx-specific differences. Figure 8 shows intercellular diffusion of NBD-M-TMA+ for a pair of Rin-37 cells. Like the experiment shown in Fig. 5B, a second electrical buzz delivered at 5 min increased k1 with no impact on the determination of k2. The rate of intercellular diffusion determined for this pair, 0.021 min1, was >10-fold lower than the rate constants determined for either Rin43 cell pair shown in Fig. 5 (0.52 and 0.75 min1), despite comparable levels of gj (7.6 nS for the Rin37 pair vs. 4.97 and 13.78 nS for the Rin43 pairs). The rate constants for four such cell pairs in which gj was >1 nS ranged from 0.014 to 0.039 min1.
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To define the selectivity of a channel, its permeability to different solutes must be contrasted (16). Junctional permeability and gj are measures of dye vs. ion movement through the channels that collectively compose a junction. If all channels in Cx43 junctions had similar NBD-M-TMA+ permeability, unitary conductance, and open probability, then regardless of their number, the selectivity (k2/gj) of all junctions would be the same. With this in mind, we calculated the selectivity for the 26 Rin43 and 4 Rin37 junctions examined in this study and found that NBD-M-TMA+ selectivity was quite variable for both (Rin43, 0.00640.906 min1·nS1; means ± SE, 0.12 ± 0.036 min1·nS1; Rin37, 0.0030.007 min1·nS1, means ± SE, 0.005 ± 0.001 min1·nS1). These results suggest the NBD-M-TMA+ selectivity of Cx43 and possibly Cx37 channels is not constant.
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DISCUSSION |
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The first gap junction protein, Cx32, was cloned and sequenced in 1986 (29); since that time, 20 additional members of the connexin gene family have been identified in the human genome (31). The functional significance of this molecular diversity is a major focus of current research. Although early studies (18, 19) demonstrated that mammalian gap junctions can be permeated by molecules up to 1,000 Da, given the diversity of possible channel types now recognized in mammalian cells, it seems unlikely that this generic view of gap junction permeation would hold true for all gap junctions, regardless of connexin composition. One of the first studies to address connexin-specific differences in selectivity was performed by Elfgang et al. (11), who examined the permeability characteristics of several gap junction channel types using an array of fluorescent dyes. Although they concluded that there were connexin-specific differences related to a combination of permeant size and charge, the study design precluded quantitative comparisons across connexins. Subsequent studies performed in a number of laboratories have led to the current position that determinants of permeability include molecular size, charge, and shape as well as channel conformation (5) and composition. Some quantitative Cx-specific comparisons of selectivity have been made (33, 38), but long experimental durations and the many assumptions required in data analysis cloud the quantitative nature of the conclusions drawn from these experiments. Furthermore, these techniques do not lend themselves to assessment of spontaneous or regulated changes of selectivity. The strategy described herein circumvents many of the limitations encountered in the previous studies.
Our approach to quantifying junctional selectivity first involves determining the rate constant for intercellular dye diffusion (k2) and then determining gj. That a single rate constant defines intercellular dye diffusion indicates the constancy of junctional permeability during the recording period and validates a single measurement of gj after the diffusion measurements. For the calculated junctional selectivity (k2/gj) to be valid, a number of conditions must be met. First, the detected fluorescence intensity must be related linearly to the concentration of free dye. We demonstrated in Fig. 1 that fluorescence and NBD-M-TMA+ concentration were indeed linearly related. That fluorescence of intracellularly located dye represents free dye is indicated by the rapid loss of fluorescence when the cells membrane is permeabilized (Fig. 1D). Second, the data acquisition rate and mathematical model must be able to distinguish k2 values that span the biologically relevant range. In Fig. 3, we show that k2 values that varied by more than three orders of magnitude resulted in predicted time courses for recipient cell filling that were readily discerned, regardless of donor cell-filling rate. Actual data and the associated RMS assessments of fit quality (see, e.g., Fig. 5, bottom) further support the adequacy of our data acquisition and fitting procedures for determining k2. Third, the methodology must be capable of detecting alterations of k2 that might occur during the course of the experiment. We show in Fig. 7 that intentional manipulation of coupling with halothane was readily detected using our protocol. This observation validates the conclusion that when the data were fit well by a single rate constant, the junctions permeability was constant during the measurement period. Fourth, an accurate measure of gj is essential. With traditional patch-clamp (CSEVC) amplifiers, high series resistance can compromise accurate measurement of gj. For example, if series resistance is 10 M, then gj would be underestimated by 10% for a 100-M
(10 nS) junction: As either series resistance or gj increases, the extent of gj underestimation also increases. We show in Fig. 2 that using switching-clamp (DSEVC) amplifiers and patch-type microelectrodes, the voltage (and therefore gj) was not detectably affected when the compensation for series resistance (in this case, 10 M
) was removed. Similar results were obtained when series resistance (same cell pair) was overcompensated to 100 M
(data not shown). Fifth, measurement of net dye transfer after a significant period of time [e.g., 30 min in the study by Valiunas et al. (33) or multiple hours in the study by Weber et al. (38)], and comparison to a single gj measurement at that time point (or an average of pre- and post-dye transfer measurements) presumes constancy of junctional permeability throughout the long measurement period (while providing no evidence for the assumption) and increases the risk that one or both parameters (permeability or conductance) might change during the experiments time course. In most of our experiments, the fluorescence data acquired in the first 10 min proved to be more than sufficient to quantify k2 and to demonstrate the constancy of the diffusion rate during that period. The measurement of gj at the conclusion of a period in which dye permeation was demonstrated as constant increased the likelihood that the measured gj was indicative of the real gj during the permeation period. Thus our approach lends itself to quantitative comparisons of the charge- and size-dependent aspects of junctional selectivity in a connexin-specific manner.
We have emphasized the suitability of our approach to the study of junctions whose behavior is stable, which facilitates comparison of data across junction types. However, our approach is also amenable to determining the acute and intermediate effects of specific treatments on junctional selectivity. We show in Fig. 7 that application of halothane during the k2 assessment period produced a readily detected decrease in k2. That k2 did not return to prehalothane values most likely reflects inadequate washout of the halothane; however, it is also possible that the contribution of a small number of channels with high NBD-M-TMA+ selectivity to the junctions selectivity was reduced or lost or that the channels were rendered less selective by halothane treatment. Distinguishing these possibilities requires additional studies that make use of perforated patch technology such that gj can be monitored continuously before, during, and after halothane application. It is worth noting that the DSEVC amplifiers can faithfully clamp the voltage of a cell despite series resistances as high as 100 M (see Fig. 2 and associated discussion); thus the increased series resistance of a perforated patch would not be a problem in such experiments.
Comparison of Strategies for Evaluating Selectivity
Two recent studies have made quantitative, connexin-specific assessments of junctional selectivity using fluorescence probes (33, 38). In both cases, the flux of dye molecules per channel per second was calculated for Cx43-composed junctions. In the first of these studies (33), the Lucifer yellow (LY) flux for the main state of Cx43 was determined to be 780 molecules·channel1·s1 at a dye concentration of 1 mM (33). In the second study (33), a value of 300,000 molecules·channel1·s1 (1 mM dye concentration) was obtained for a dye (Alexa 488) similar to LY in size and charge.
The usefulness of such calculations depends on the validity of the underlying assumptions. In the Valiunas et al. (33) study, which explored Cx43 junctions in HeLa cells, these assumptions were 1) the volume of both cells equaled 1 pL; 2) all dye in the donor cell was unbound; 3) at steady-state fluorescence, dye concentration in the donor cell equaled that in the pipette; 4) intercellular dye diffusion was mediated by the full open channel; 5) macroscopic conductance measurements were not compromised by series resistance; 6) the entire macroscopic conductance was contributed by full open channels; 7) channel open probability was equal to 1; and 8) net flux per unit time was constant (typical experiment duration 2050 min). Two of these assumptions are of particular concern. First, their calculations depend on accurate assessment of dye concentration in donor and recipient cells (rather than determining a rate constant that is independent of concentration). The fact that LY concentrates in the nucleus of cells means that the detected fluorescence is not representative of the concentration of free dye. In addition, the cell volume could be different from 1 pL. Second, the assumption of constant net flux per unit time during experiment durations approaching 1 h is not warranted for two reasons. First, the concentration difference between the cells changes during the measurement period; second, the selectivity of the junction may not be constant. Determining a rate constant eliminates these problems. These assumptions would lead to underestimation of flux per channel. Although not explicitly stated in the Valiunas et al. (33) study as an assumption, if open states other than the full open state were better permeated by the dye, then their contribution to intercellular flux would be missed and instead would be attributed to the full open state. This could lead to an overestimation of the flux per channel per second for the full open state and an underestimation of flux for other open states. In the second study, Weber et al. (38) estimated the selectivity of Cx43 junctions as formed by pairs of frog oocytes with a series of Alexa dyes (of graded size). In addition to the impact of many of the assumptions mentioned above, the large cell size, the significant binding of dye to cytosolic components, and the disparity between the thickness of the microscopes focal plane and the thickness of the oocyte presented technical challenges that were addressed by modeling intracellular diffusion of the various dyes (28) and monitoring transjunctional diffusion for a long period of time (6 h). Given the many assumptions in these two studies, it is not clear which of the disparate values for flux per channel per second more closely approximated the real value for the full open state of the Cx43 channel.
For purposes of comparison with these previously published studies, we calculated the flux per channel per second for NBD-M-TMA+ for channels in high- vs. low-selectivity junctions. This calculation requires the rate constant (expressed in s1), the number of channels mediating the flux (by solving the equation gj = N·Po·j for N, assuming an open probability of 1.0 and using the experimentally derived unitary conductance), and a cell volume such that the number of molecules available to flux through the channel can be calculated for any desired dye concentration. In the experiment shown in Fig. 5B, the rate constant was 0.75 min1 (or 0.0125 s1) and gj was 13.78 nS. Although brief segments of the single-channel recording provided evidence for a single population of channels (see Fig. 6C), the amplitude histogram derived from the entire recording for this pair (with 235 events; data not shown) revealed events with amplitudes ranging from 60 to 135 pS, with the bulk of events occurring between 70 and 95 pS. Thus the number of channels participating in the flux was between 145 and 197 channels. The relative selectivity for NBD-M-TMA+ of these open states is not known; but assuming equal selectivity and a cell volume of 1 pL, the flux per channel would be between 38,200 and 51,900 molecules·channel1·s1 with a dye concentration of 1 mM. Similar calculations for the highest vs. lowest selectivity junctions (26 Rin43 cell pairs) revealed a range from 3,560 to nearly 1 x 106 molecules·channel1·s1 for Cx43 channels and from 4,565 to 10,895 molecules·s1·channel1 for Cx37 channels (1 mM dye concentration). Interestingly, for two pairs of cells in which the amplitude histograms revealed a single population of channels with conductances of 80100 pS, the channel flux rates were 12,700 and 16,000 molecules/s.
The range of channel flux rates observed in our study for NBD-M-TMA+ encompassed both of the previously published values (33) and expanded them somewhat (possibly reflecting the positive charge of NBD-M-TMA+ and/or its smaller size). It is interesting to consider the possible basis for such variability. Of the many assumptions necessary to make these calculations, the assumption that all Cx43 channels share a similar selectivity for the probe molecules is the most tenuous, and, if incorrect, it could explain the variability reported in all three studies. The linear relationship between relative dye intensity and gj reported by Valiunas et al. (33) is consistent with the conclusion that all channels in Cx43 junctions share a common (LY) selectivity. The data are also consistent, however, with a mixed population of channels in which only a subset is permeated by the dye. Several observations suggest this might be the case. First, Bukauskas et al. (5) found that the full open state of the channel was nonselective for anions vs. cations, whereas the voltage-induced substrate was more anion selective and size selective. Second, the LY flux rate reported by Valiunas et al. is comparable to the NBD-M-TMA+ flux rate reported herein for the few cell pairs in which only full open channels were apparent. Thus the lower per channel flux rates may correspond to the full open channel. Certainly for Rin43 pairs, it appears that a diversity of channel amplitudes give rise to a diversity of junctional selectivity values. Because j varies with the phosphorylation of the channel proteins (24, 26), the implications of our findings are that selectivity is a regulated function of Cx43 channels and that the selectivity of a Cx43 junction depends on the contribution to the junction of well-permeated vs. poorly permeated (differentially phosphorylated) channels.
The molecular shape of the potential permeant (in addition to its size and charge) may also influence channel permeation. Bevans et al. (4) showed that Cx32 homomeric connexins allow the diffusion of negatively charged cAMP and cGMP, but Cx32-Cx26 heteromeric connexins restrict the passage of cGMP but not cAMP. Other studies have shown that Cx32 channels are 10-fold more efficient than Cx43 channels in mediating diffusion of adenosine; as phosphates are added to the adenosine molecule to form, successively, AMP, ADP and ATP, however, Cx43 becomes the better mediator of diffusion. Thus ATP diffuses through Cx43 channels 300 times more efficiently than through Cx32 channels (13). The simplest explanation of such results is that there is a binding-type interaction between the permeant and the channel that facilitates discrimination of otherwise similar molecules.
In summary, our strategy for determining junctional selectivity reduces the number of assumptions needed to evaluate Cx- or treatment-specific effects on junctional selectivity. Although calculations of flux per channel per second are possible, it is the net flux through the collection of channels that determines the success of coordinated function in a population of cells. Although previous estimates of these flux rates have left some in doubt regarding the role that gap junctions could play in mediating the spread of short-lived chemical signals (33), the flux rates observed in the present study and those of Weber et al. (38) strongly suggest that such a role is not only possible but likely. By quantifying intercellular dye diffusion as a rate constant, the necessity of long experiment duration (required for the donor cell to achieve a steady state and thereby a predictable dye concentration) is eliminated. Furthermore, changes in rate constant are detected easily; therefore, the absence of change in rate constant justifies the assumption of constant junctional properties during the course of the experiment. Finally, by using a dye with little or no tendency to bind to intracellular components, the assumption that fluorescence is indicative of diffusible dye is also warranted. Our strategy should facilitate the quantitation of connexin-specific and treatment-induced differences in junctional selectivity. Experiments making use of different dyes to compare wild-type and mutant connexins should facilitate the determination of whether selectivity is a regulated function of Cx43 channels and, if so, should be able to determine the signaling events involved in that regulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Samples of NBD-M-TMA+ can be obtained by contacting Dr. Janis Burt (jburt{at}u.arizona.edu) or purchased from Dr. Gene Mash in the Department of Chemistry (emash{at}u.arizona.edu) or Dr. Stephen Wright in the Department of Physiology (shwright{at}u.arizona.edu) at the University of Arizona.
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REFERENCES |
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