Gap junctional coupling in lenses from alpha(8) connexin knockout mice

Abstract

Lens fiber cell gap junctions contain alpha(3) (Cx46) and alpha(8) (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either alpha(3) or alpha(8) connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from alpha(3) connexin knockout lenses. Here, we present original data from alpha(8) connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell-cell contact of approximately 1 S/cm(2), which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is approximately 0.4 S/cm(2), and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/-) was approximately 70% of normal and 30-40% of the normal for both -/- lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of alpha(3) +/- lenses. In contrast, the coupling of MF in the alpha(8) +/- lenses was the same as normal. Moreover, no coupling was detected in the MF of alpha(3) -/- lenses. Together, these results suggest that alpha(3) connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and alpha(3) connexin -/- lenses. However, in alpha(8) -/- lenses, the pure alpha(3) connexin junctions did not gate closed in the response to acidification. Since alpha(3) connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears alpha(8) connexin regulates the gating of alpha(3) connexin. Both connexins are clearly important to lens physiology as lenses null for either connexin lose transparency. Gap junctions in the MF survive for the lifetime of the organism without protein turnover. It appears that alpha(3) connexin provides the long-term communication in MF. Gap junctions in DF may be physiologically regulated since they are capable of gating when the cytoplasm is acidified. It appears alpha(8) connexin is required for gating in DF.

Figure 1

The cellular structure of the lens. Based on transport properties, the lens can be divided into three radial zones. The surface (S) includes the basal lateral membrane of the anterior epithelium and the basal membrane of differentiating fibers (DF) cells in contact with the posterior surface. These membranes have a relatively high permeability for K⁺ and perform nearly all of the active transport in the lens. The DF and mature fibers (MF) are in communication with each other and with the surface membranes through an extensive network of gap junctions. However, the functional and structural properties of gap junctions coupling DF and MF differ, so these two domains are studied separately.

Figure 2

Immunoblot of the α₈ connexin knockout mice. The NaOH-insoluble protein fractions were prepared from the lenses of +/+, α₈ +/−, and α₈ −/− mice at the age of 8 wk (Gong et al. 1997). Immunoblot analysis was performed using antibodies against α₈ connexin (top), α₃ connexin (middle), and MP26 (bottom), respectively. Equal portions of the samples (∼15 mg total proteins) were loaded in each lane.

Figure 3

Resting voltages. The value of the resting voltage is plotted as a function of normalized distance from the lens center (r/a), where r (in centimeters) is the distance from the center and a (in centimeters) is the lens radius. The points represent data from lenses of +/+ and α₈ −/− mice. The solid smooth curves represent the expected distribution of resting voltages based on a model of the circulating current that flows into the lens along extracellular spaces, crosses the fiber cell membrane, and then flows toward the surface of the lens from cell to cell via gap junctions (for review see Mathias et al. 1997). The dashed line is an arbitrary fit to resting voltage data from lenses of α₃ −/− mice (Gong et al. 1998). The α₈ −/− lenses are depolarized at all locations, suggesting some indirect effects on surface cell membrane permeability. In contrast, the α₃ −/− lenses have a normal voltage in DF, but the MF depolarize in a way that reflects loss of gap junctional coupling.

Figure 4

The cumulative series resistance (R_S)_. The closed circles in A–C are R_s data from six lenses from +/+, +/−, and −/− α₈ connexin knockout mice. The solid lines represent the best fit using and with a = 0.101 cm for +/+ and +/− lenses, a = 0.08 cm for −/− lenses, b = 0.8a, and with the values of R_DF and R_MF listed in each panel. The best fits are graphed together in D. The lenses from α₈ −/− mice have many abnormalities that are secondary consequences of the knockout of α₈ connexin. The lenses from α₈ +/− mice are normal except for changes in coupling conductance. In these lenses, removal of ∼50% of α₈ connexin significantly increases R_DF, but has no effect on R_MF, suggesting the α₈ connexin may not be a functional component of gap junctions coupling MF.

Figure 5

pH-sensitive gating. A compares the coupling of DF from wild-type and α₈ connexin −/− lenses when superfused with Tyrode bubbled with 100% CO₂. The CO₂-saturated solution was superfused during the time period indicated by the bar. The initial values of R_P. R_DF for the two −/− lenses was 5.9 and 10.1 KΩ-cm, whereas the initial value of R_P in the +/+ lens was 3.4 KΩ-cm, and all of these values were stable before CO₂ exposure. To facilitate comparison, each resistivity was normalized to its initial value, and then graphed as a function of time. In a 10-min exposure to 100% CO₂, the R_P of DF in the wild-type lens increased 4.7-fold. This was a typical response, similar to the responses we recorded from a number of lenses from wild-type mice of different litters. In contrast, R_P of the DF in α₈ −/− lenses increased only 1.6-fold. Thus, in the α₈ −/− lens, homotypic channels made from α₃ connexin have little or no sensitivity to pH. The surface cells express α₁ (Cx43) connexin, so the limited pH gating seen in A could be due to α₁. B compares the cumulative series resistance (R_S in ) in lenses from +/+ and α₈ −/− mice. Again, the CO₂ exposure was during the period indicated by the bar and before t = 0, the values of R_S were stable. As illustrated by , this resistance is dominated by the contribution of MF. Again, the resistances were normalized to their initial values, and then graphed as a function of time exposed to 100% CO₂. The +/+ data were mean ± SD from four lenses. Although the MFs of +/+ lenses are not sensitive to pH, DFs are sensitive, hence, R_S increases significantly. The α₈ −/− data are mean ± SD from three lenses. In the α₈ −/− lenses, all fiber cell gap junctions contain homotypic channels made up of α₃ connexin. If the small increase in resistance of DF from α₈ −/− mice shown in A is confined to DF or surface cells, it will be a very small fraction of the total R_S shown in B. Indeed, the total R_S of α₈ −/− mice shows no significant increase on exposure of the lenses to 100% CO₂.