Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures

Abstract

We have developed a simple dye transfer method that allows quantification of the gap-junction permeability of small cultured cells. Fluorescent dyes (calcein and Lucifer yellow) were perfused into one cell of an isolated cell pair using a patch-type micropipette in the tight-seal whole cell configuration. Dye spreading into the neighboring cells was monitored using a low-light charge-coupled device camera. Permeation rates for calcein and Lucifer yellow were then estimated by fitting the time course of the fluorescence intensities in both cells. For curve fitting, we used a set of model equations derived from a compartment model of dye distribution. The permeation rates were correlated to the total ionic conductance of the gap junction measured immediately after the perfusion experiment. Assuming that dye permeation is through a unit-conductance channel, we were then able to calculate the single-channel permeance for each tracer dye. We have applied this technique to HeLa cells stably transfected with rat-Cx46 and Cx43, and to BICR/M1R(k) cells, a rat mammary tumor cell line that has very high dye coupling through endogenous Cx43 channels. Scatter plots of permeation rates versus junctional conductance did not show a strictly linear correlation of ionic versus dye permeance, as would have been expected for a simple pore. Instead, we found that the data scatter within a wide range of different single-channel permeances. In BICR/M1R(k) cells, the lower limiting single-channel permeance is 2.2 +/- 2.0 x 10(-12) mm3/s and the upper limit is 50 x 10(-12) mm3/s for calcein and 6.8 +/- 2.8 x 10(-12) mm3/s and 150 x 10(-12) mm3/s for Lucifer yellow, respectively. In HeLa-Cx43 transfectants we found 2.0 +/- 2.4 x 10(-12) mm3/s and 95 x 10(-12) mm3/s for calcein and 2.1 +/- 6.8 x 10(-12) mm3/s and 80 x 10(-12) mm3/s for Lucifer yellow, and in HeLa-Cx46 transfectants 1.7 +/- 0.3 x 10(-12) mm3/s and 120 x 10(-12) mm3/s for calcein and 1.3 +/- 1.1 x 10(-12) mm3/s and 34 x 10(-12) mm3/s for Lucifer yellow, respectively. This variability is most likely due to a yet unknown mechanism that differentially regulates single-channel permeability for larger molecules and for small inorganic ions.

FIGURE 1

Structure of the two fluorescent tracer dyes Lucifer yellow (a) and calcein (b).

FIGURE 2

Schematic representation of the three-compartment model for dye transfer in a perfused cell pair.

FIGURE 3

Camera calibration curves for calcein (a and b) and Lucifer yellow concentration (c and d). (a and c) Uncalibrated gray-scale intensities for series dilutions of the two tracer dyes. The nonlinearities due to the camera's gamma correction are clearly visible. (b and d) The same series after correction by the densitometric calibration facility of the image analysis program.

FIGURE 4

Time course of a calcein perfusion experiment in BICR/M1R_k cells. (a) Phase contrast and fluorescence images taken at 100-s intervals from a series. (b) Evolution of a gray-scale profile taken across the cell pair along the dashed line shown in the phase-contrast image in a. Gray-scale values were corrected as shown in Fig. 3 d. Equilibration of cell 1 with the perfusion pipette is fast and almost complete within the first two frames shown. There is also no evidence for a diffusion profile within cell 2, indicating that cytoplasmic diffusion is not rate-limiting.

FIGURE 5

Time course of the integrated fluorescence intensities for both cells of a perfused cell pair for calcein (a) and Lucifer yellow perfusion (b). The solid lines indicate the fits of the diffusion model to the data. The permeation rates are κ_pip = 19.6 ± 1.5 μm³/s and κ_j = 3.64 ± 0.09 μm³/s for the calcein perfusion experiment (a) and κ_pip = 9.0 ± 0.3 μm³/s and κ_j = 2.09 ± 0.04 μm³/s for the Lucifer yellow perfusion experiment. Note that the permeabilities are of similar magnitude, although the time course for Lucifer yellow appears slower due to the larger cell volumes in this pair (cell₁ = 860 fl and cell₂ = 1050 fl in a versus cell₁ = 2650 fl and cell₂ = 1900 fl in b). Also note that the fluorescence integral measures the total amount of tracer within a cell. This depends on the cell volume as well as on concentration and, consequently, will not generally be equal once equilibrium has been reached if the cells are asymmetric in size.

FIGURE 6

Electrophysiological recordings for the determination of the cell-cell conductance. (a) Current and voltage recordings from both cells of a cell pair. (b) Current-voltage relation of the transjunctional current. The I/V curve was fitted with a straight line where the slope corresponds to the cell-cell conductance.

FIGURE 7

Distribution of the dye transfer rates versus cell-cell conductance for calcein in BICR/M1Rk cells shown as (a) linear and (b) double logarithmic plots. The dashed lines represent the lower-limiting slope corresponding to the peak of the single-channel permeances shown in Fig. 8, below, and a putative upper limit. Note that, due to the double logarithmic scaling in b, straight lines through the origin are transformed into parallel lines with the same slope but different offset.

FIGURE 8

Double-logarithmic plots of dye transfer rates versus cell-cell conductance and histograms of the distribution of the single-channel permeances for calcein (a–f) and Lucifer yellow (g–l) in BICR/M1Rk cells (a, d, g, and j), HeLa-Cx43 transfectants (b, e, h, and k), and HeLa Cx46 transfectants (c, f, i, and l). The dashed lines in the scatter plots represent the lower-limiting slopes corresponding to the peaks of the corresponding distribution of single-channel permeances shown below. Note that, due to the double logarithmic scaling, straight lines through the origin are transformed into parallel lines with the same slope but different offset. Parameters for the slopes and distributions are given in Table 1.

FIGURE 9

Dependence of single-channel permeances on tracer size (left column) and charge (right column) for BICR/M1R_k cells (a and b), HeLa-Cx43 transfectants (c and d), and HeLa-Cx46 transfectants (d and e). The gray bars represent logarithmic histograms of the distribution of single-channel permeances, with the horizontal width of a bar corresponding to the relative frequency and the vertical width to the bin width of 0.2 log₁₀ units, respectively. The gray lines correspond to the single-channel permeances predicted by a simple model for a straight cylindrical pore of radius 0.74 nm (solid line), 0.86 nm (dashed line), and 1.19 nm (dotted line). The lower bounds of our single-channel permeances are roughly consistent with a simple pore of ∼1 nm radius. For reference, we have included data reported by Weber et al. (21) for Alexa-dyes in Xenopus oocytes (□) and by Valiunas et al. (23) and Biegon et al. (15) for N2A- (▵) and Novikoff hepatoma cells (▿) expressing Cx43 to the BICR/M1R_k and HeLa-Cx43 data sets. Note that for both Lucifer yellow and calcein the range of permeances comes close to the Xenopus data, although the maximum values still fall short by a factor of 5–10.