Properties of connexin 46 hemichannels in dissociated lens fiber cells

Abstract

Purpose: To characterize the properties of connexin 46 hemichannels in differentiating fiber cells isolated from mouse lenses.

Methods: Differentiating fiber cells were isolated from mouse lenses using collagenase. Cellular localization of connexin 50 (Cx50) and connexin 46 (Cx46) was assessed by immunofluorescence. Membrane currents were recorded using whole cell patch clamping. Dye uptake was measured using time-lapse imaging.

Results: In freshly dissociated fiber cells isolated from knockout Cx50 (KOCx50) mouse lenses, removal of external divalent cations induced a macroscopic current composed of large conductance channels. This current was reduced at a holding potential of -60 mV, activated on depolarization, and had a reversal potential near 0 mV. These properties were very similar to those of Cx46 hemichannel currents recorded in single Xenopus oocytes. If the currents observed in divalent cation-free Ringer's solution were due to Cx46 hemichannel opening, then dye influx by gap junctional/hemichannel permeable dyes should be measurable in the fiber cells. To measure dye influx, the authors used the positively charged dyes, propidium iodide (PrI) and 4'-6-diamidino-2-phenylindole (DAPI). In the absence of external calcium, fiber cells took up both dyes. Furthermore, dye influx could be inhibited by hemichannel blockers. To confirm that this current was due to Cx46 hemichannels, the authors studied fiber cells isolated from the lenses of double knockout (Cx46(-/-); Cx50(-/-)) mice and demonstrated that both the calcium-sensitive conductance and dye influx were absent.

Conclusions: These results show that Cx46 can form functional hemichannels in the nonjunctional membrane of fiber cells.

Figure 1.

Membrane currents in a KOCx50 fiber cell. (A) A family of current traces was recorded from the fiber cell shown in (B) using the whole cell patch clamp technique. A series of depolarizing steps was applied in 10 mV increments between −60 and 80 mV from a holding potential of −80 mV. (C) I–V relations obtained from the data shown in (A). The current was measured at the end of the 8-second pulse and plotted as a function of voltage after correction for liquid junction potential.

Figure 2.

The amplitude of the hemichannel current is linearly correlated to membrane capacitance. (A) Steady state current amplitude (measured at the end of an 8-second pulse to 30 mV) is plotted as a function of membrane capacitance. n = 22. (B) Plot of cell length versus membrane capacitance for same data set shown in (A).

Figure 3.

(A) A family of whole cell tail currents recorded from a lens fiber cell. The voltage clamp protocol is shown. The voltage was stepped from a holding potential of −60 mV to 80 mV for 1.5 seconds to open the hemichannels and then repolarized to voltages between −60 and +10 mV. (B) Instantaneous I–V relationship determined by measuring the isochronal tail currents. Continuous line represents the best fit of the instantaneous I–V relationship to a linear regression. (C) Time constants of deactivation plotted as a function of voltage. The time constants of deactivation were obtained by fitting the time course of deactivation of the tail currents shown in (A) to a single exponential (solid light gray lines).

Figure 4.

Whole cell current traces recorded from a small fiber in response to voltage clamp steps to the indicated potentials from a holding potential of −60 mV. The whole cell current shows discrete steps indicative of opening and closing of single hemichannels. The fiber cell had a length ∼20 μm, based on its membrane capacitance of 13.2 pF.

Figure 5.

Effect of divalent cations. (A, B) Families of current recorded from a lens fiber cell before (A) and after (B) application of divalent cations. The voltage clamp protocol consisted of sequential steps from a holding potential of −60 mV to 50 mV in 10 mV increments. The membrane potential was continuously held at −60 mV between the two trials. (C) Isochronal I–V relationship (measured at the end of the pulse) before (open circles) and after (solid triangles) application of divalent cations.

Figure 6.

Dye uptake in a differentiating fiber cell isolated from the lens of a knockout Cx50 mouse. (A) Hoffmann image of the fiber cell used in the dye uptake experiment. (B) PrI images at different time points of a time-lapse recording. The cell was exposed to PrI (2 μM) starting at time 0. (C) Dye uptake was decreased by raising [Ca²⁺]_o. To measure changes in the rate of dye uptake over time, the integrated fluorescence intensity from the ROI shown in (B) is plotted as a function of time in arbitrary units (a.u.). The cell was initially bathed in nominally divalent cation-free solution. At the time indicated by the first arrow, the cell was exposed to a solution containing 2 mM [Ca²⁺]_o. Calcium was washed out at the time indicated by the second arrow. All the solutions contained PrI (2μM).

Figure 7.

The rate of DAPI and PrI uptake are reduced by agents known to block connexin hemichannels. (A–E) Selected examples that illustrate changes in rate of DAPI and/or PrI uptake by KOCx50 fiber cells after exposure to 2 mM calcium (A, B), 250 μM gadolinium (C), 300 FFA μM (D), or 3 mM octanol (E). (F) Averaged data of effects (normalized as percent of rate of uptake in control solution) of calcium, FFA, gadolinium, and octanol on rate of uptake of DAPI (dark gray bars) and PrI (light gray bars) by KOCx50 fibers. The number of cells is indicated in parentheses.

Figure 8.

Immunolocalization of Cx46 and Cx50 in lens fiber cells collected from adult knockout mice. (A) Cx50 localizes along the plasma membrane between lens fiber cells from KOCx46 mice. (B) Cx46 localizes along the membranes between lens fiber cells from KOCx50 mice. (C, D) Lens fiber cells from Cx46, Cx50 double KO mice, shown by phase microscopy, exhibit no immunoreactivity for Cx46 or Cx50. Regions of plasma membrane apposition are indicated by asterisks and DAPI-stained nuclei are seen in all merged images (A, B, D).

Figure 9.

Loss of Cx50 and Cx46 abolishes calcium-sensitive membrane currents in single fiber cells. (A) Example of membrane currents recorded from a double KO fiber in divalent cation-free NaGluconate Ringer's solution. The voltage clamp protocol consisted of sequential steps from a holding potential of −60 mV to 80 mV in 10 mV increments. Dashed line: zero current. (B) I–V relations obtained from the data shown in (A). The current was measured at the end of the 8-second pulse and plotted as a function of voltage. (C) Steady state current (measured at the end of an 8-second pulse to 80 mV) plotted as a function of membrane capacitance for the double KO cells (solid circles) and the KOCx50 cells (open squares). (D) Histogram compares the mean area-specific current of the double KO cells (open bar) and the KOCx50 cells (black bar). The area-specific current was calculated from the data shown in (C), assuming an area-specific membrane capacitance of 1 μF/cm². Number of cells is indicated in parentheses.

Figure 10.

PrI uptake is reduced in fibers isolated from double KO lenses. The kinetics of PrI uptake by KOCx50 cells (open circles) and double KO cells (solid circles); each curve is the average of the fluorescence intensity from several ROI positioned on different fiber cells plotted as a function of time (normalized to fluorescence intensity at T = 0). The cells were initially bathed in nominally divalent cation-free solution. At the time indicated by the arrow, the cells were exposed to a solution containing 2 mM [Ca²⁺]_o. Number of cells is indicated in parentheses.