Correspondence to: Richard T. Mathias, Department of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794-8661. Fax:(631) 444-3432 E-mail:rtmathias{at}physiology.pnb.sunysb.edu.
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Abstract |
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Lens fiber cell gap junctions contain 3 (Cx46) and
8 (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either
3 or
8 connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In
3 connexin knockout lenses. Here, we present original data from
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 Figure 1, DF) has an average coupling conductance per area of cellcell contact of
1 S/cm2, which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see Figure 1, MF), the average coupling conductance is
0.4 S/cm2, 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
70% of normal and 3040% 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
3 +/- lenses. In contrast, the coupling of MF in the
8 +/- lenses was the same as normal. Moreover, no coupling was detected in the MF of
3 -/- lenses. Together, these results suggest that
3 connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and
3 connexin -/- lenses. However, in
8 -/- lenses, the pure
3 connexin junctions did not gate closed in the response to acidification. Since
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
8 connexin regulates the gating of
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
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
8 connexin is required for gating in DF.
Key Words:
pH gating, coupling conductance, 8 connexin Cx50,
3 connexin Cx46
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INTRODUCTION |
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The lens is a multicellular syncytial tissue that has several structurally and functionally distinct domains. A single layer of epithelial cells covers the anterior hemisphere (see Fig 1). At the equator, the epithelial cells begin to differentiate and elongate into fiber cells, which are laid down in shells to form the bulk of the lens mass. Thus, the oldest fiber cells are near the lens center, and the youngest are at the periphery. The fiber cells are flattened hexagons in cross section, with dimensions of 3 x 9 µm (see Fig 1). They extend from the anterior to posterior sutures, where they are bound together. The fiber cells pack closely together with the extracellular spaces of
10 nm in width, thus, the volume of extracellular space is
200 times smaller than the volume of intracellular space. The surface to volume ratio of a fiber cell is
6,000 cm-1, so a small mouse lens of diam 0.2 cm has
25 cm2 of fiber cell membrane, whereas the outer surface of such a lens has an area of
0.13 cm2. In the lenses of larger mammals, this disparity in areas is much larger.
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The lens is the only transparent tissue that is more than a few cell layers thick. Its large size and need for transparency have posed some unique problems that have led to interesting physiological solutions, many of which appear to involve fiber cell gap junctions. Consider the problems: tissues the size of the lens are generally vascularized to enable access of metabolites to interior cells. However, blood vessels absorb and scatter light, so the lens is avascular. Thus, interior cells of the lens are a long distance from a source of metabolites. Intracellular organelles also scatter light and make most cells translucent, hence, the majority of lens cells, the mature fibers (see Fig 1, MF), have disposed of their organelles. These cells cannot divide or make new protein, so gap junctions and other proteins must survive for the life of the organism without renewal. Finally, to focus images on the retina, the lens needs a relatively high index of refraction, which is a property of the cytoplasm of lens fiber cells. However, the extracellular spaces between fiber cells of the lens have a lower index of refraction, so their width must be less than the wavelength of visible light, otherwise they reduce transparency. This requires that lens cells precisely maintain a constant volume, since a 10% reduction in intracellular volume would result in about a 20-fold expansion in extracellular volume and cause light scattering.
A characterization of functional properties of lens cells (for review see
Fiber cells lack Na/K ATPase activity and have relatively low conductance membranes that appear to have lost their K+ channels. The cells have a small Na+ conductance per unit area of membrane, but because of the relatively large area of fiber cell membrane in a lens, the total fiber cell Na+ conductance is significant. Because the fiber cells lack functional Na/K pumps and K+ channels, if they are isolated in physiological saline, they do not maintain a normal negative resting voltage and swell until they burst (
Fiber cell gap junctions contain connexins 3 (Cx46) and
8 (Cx50). The functional and structural properties of these gap junctions change abruptly at the border between differentiating fibers (Fig 1, DF in blue) and mature fibers (Fig 1, MF in green). In the peripheral shell of DF, large gap junctional plaques, containing both
3 and
8, occur primarily on the broad sides of equatorial fiber cells (
3 and
8 connexin in the DF to MF transition, but not due to synthesis of new proteins since fiber cells lack the machinery necessary for protein synthesis. The coupling conductance of MF is somewhat lower than the average value in DF, it is relatively uniform throughout the core of MF, and it is insensitive to changes in pH (for review see
The most dramatic physiological property of a normal lens is the steady-state current that is illustrated by the lines of flow in Fig 1. This current is large by epithelial standards, reaching 30 µA/cm2 at either pole or the equator, it is present in lenses from all species studied, and it flows around and through the lens. Thus, one has to believe it is critical for lens homeostasis. Our hypothesis (
Although we have hypotheses on the roles of fiber cell gap junctions in lens physiology, before studies of lenses from the connexin knockout mice, the purpose of the two connexins, 3 and
8, was not known. Both connexins were thought to be present in junctions coupling MF and DF, however, as mentioned above, MF and DF junctions are structurally and functionally different. In our previous study (
3 connexin caused complete uncoupling of MF. Although this demonstrated that
3 connexin is necessary for coupling of MF, it did not rule out the presence of functional
8 connexin in the MF junctions. Moreover, this previous study provided no clues on the purpose of
8 connexin. To better understand the roles of both
3 and
8 connexins in MF and DF junctions, we have produced genetically engineered mice, which lack the gene coding for
8 or
3 connexin. This paper describes the functional properties of gap junctional coupling in lenses lacking
8 connexin and compares these results with those of our previous study (
3 connexin.
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MATERIALS AND METHODS |
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Generation of 8 Connexin Knockout Mice
The mouse 8 connexin gene was cloned from a 129Sv mouse genomic library by using the mouse
8 connexin gene as a probe (
8 connexin knockout mice was similar to that described previously for the generation of the
3 connexin knockout mice (
8 connexin knockout mice will be more fully described in a subsequent paper. The
8 connexin mice used in this study were of a mixed background strain (C57 x 129Sv).
Immunoblot Analysis of Lens Homogenates
NaOH-insoluble fractions of lens homogenates from wild-type +/+, heterozygous +/-, and homozygote -/- 8 connexin knockout mice were prepared and analyzed using 10% SDSpolyacrylamide gels, as previously described (
8 connexin antibody (Alpha Diagnostics) was used at a dilution of 1:500, and a rabbit polyclonal antibody to MIP (1:1,000) and to
3 connexin (1:200) were used for immunoblot analysis (
Electrophysiology
Because of litter to litter variation in the coupling of wild-type mouse lenses (8 +/-, and
8 -/- mice from one litter. In this litter, there were six lenses from each class. Each mouse was killed by intraperitoneal injection of sodium pentobarbitone (57.1 µg per gram body weight), and then its lenses were immediately dissected from the eyes and used for electrophysiological experiments. The dissection and other experimental procedures are identical to those described in
i) was recorded. The induced voltage and injected current waveforms were sent to a Hewlett Packard Fast Fourier Analyzer, where the frequency domain impedance was computed in real-time. At sinusoidal frequencies above
100 Hz, the induced voltage response in the intracellular and extracellular compartments within the lens become essentially identical, and
i
IRS, where RS (
) is the cumulative series resistance between the point of voltage recording and the lens surface. RS is proportional to the depth into the lens and the parallel resistivity of the intra- and extracellular compartments; Rp (
-cm;
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(1) |
Ri(-cm) is the effective intracellular resistivity, which depends primarily on the gap junctional coupling resistance between fiber cells. Re (
-cm) is the effective extracellular resistivity due to the small tortuous extracellular clefts. In normal physiological conditions, Re >> Ri, hence Rp
Ri. In some of the experimental conditions, gap junctional conductance is greatly reduced or zero, in which case Rp = Re. Re is assumed to be approximately uniform throughout the lens, whereas Ri is lower in DF than MF. The details of the increase in Ri in the DF to MF transition cannot be resolved by our techniques, yet it is clear that the increase occurs over relatively few cell layers. For simplicity, we assume that Ri changes abruptly at the DF to MF transition. Thus, RP in a well coupled lens is approximately equal to Ri, which has the value RDF in the DF zone and RMF in the MF zone. Our initial microelectrode placement recorded RS in the DF zone and was used to determine RDF:
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(2) |
The radial location r (measured in centimeters) is the distance of the voltage recording electrode from the lens center, a (measured in centimeters) is the lens radius, and b (measured in centimeters) is the radial location of the transition from DF to MF (0.02 cm below the surface). The voltage recording microelectrode was advanced in a series of steps toward the lens center and these recordings were used to determine RMF:
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(3) |
To test the sensitivity of coupling to acidification, a measurement of RS in either DF or MF was made while superfusing the lens with a Tyrode solution that had been bubbled with 100% CO2 (
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RESULTS |
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Analysis of 8 Knockout Lenses by Immunoblotting
The 8 connexin gene was disrupted in mice using a strategy that was previously applied for generating the
3 connexin knockout mice (
8 connexin gene was determined by Southern blot and PCR analysis. Connexin expression in lenses from +/+,
8 connexin +/-, and -/- mice was estimated using Western blotting (Fig 2). Approximately the same amount of material was loaded into each lane, and the intensities of the bands were estimated using a densitometer. This analysis confirmed that the
8 connexin protein was not present in the homozygous
8 -/- mice lenses. Within the accuracy of this technique, in the lenses of heterozygous
8 +/- mice the amount of
8 protein was reduced by
50% (
3 connexin and MIP, appeared to be about the same in lenses of wild-type and
8 +/- or -/- mice. The multiple bands present in the
3 blot most likely represent different phosphorylated forms of this connexin.
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Physiological Properties
As shown by the solid circles in Fig 3, when the intracellular voltage (measured with respect to the external bathing solution) in a normal mouse lens is graphed as a function of distance from the lens center, there is a voltage gradient with the most central cells having a resting voltage that is 1015 mV more positive than the peripheral cells (for review see 8 -/- mice (Fig 3, triangles) is uniformly depolarized relative to that of wild-type lenses. The input conductance per unit area of lens surface was higher in lenses from -/- mice, with -/- GIN = 1.7 ± 0.3 mS/cm2 versus +/+ GIN = 1.4 ± 0.2 mS/cm2. The higher conductance and depolarized resting voltage in the
8 connexin knockout lenses indicate effects on transmembrane ion fluxes as well as on cellcell communication. As initially described in
8 -/- mice are abnormally small; for the lenses used in this study, the +/+ radius, a = 0.101 ± 0.001 cm versus the -/- radius, a = 0.080 ± 0.002 cm (mean ± SD, n = 6 each).
8 connexin knockout mice were due to effects during early development. Lenses from -/-
8 mice have reduced transparency (
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The regional distribution of resting voltage in the 3 versus
8 connexin knockout mice is quite different. The dashed curve in Fig 3 was generated using resting voltage data from lenses lacking the
3 connexin (
3 lenses have a normal voltage and perfectly transparent DF, but they rapidly depolarize in the transition to MF, where there is complete loss of gap junctional coupling. Hence, this distribution of resting voltage may be directly related to the loss of coupling of MF with the surface cells, which possess the lens K+ channels. However, these lenses have a dense central cataract that fills about half the volume of the MF.
Because of the general deterioration of the lenses from either 3 or
8 connexin knockout mice, it is difficult to separate direct from indirect consequences of the lack of a particular connexin. However, lenses from
8 +/- mice were normal with regard to resting voltage, and had the same input conductance as wild-type lenses (+/- GIN = 1.4 ± 0.2 mS/cm2). Similarly, the
3 +/- lenses appeared nearly normal. Thus, reductions in gap junctional conductance in either
3 or
8 connexin +/- lenses are more likely to reflect directly the reduction in the amount of connexin, rather than some secondary effect.
The Distribution of Functional 3 and
8 Connexins
Impedance measurements were conducted on lenses from wild-type and 8 knockout mice and used to generate the data in Fig 4. These data represent the cumulative series resistance (RS in Equation 2 or Equation 3) due to gap junctions coupling fiber cells between the point of recording (r centimeters from the lens center) and the surface of the lens with radius = a cm. Because of spherical geometry, the cumulative resistance increases more than linearly, and because the gap junctional coupling resistance increases in the DF to MF transition, the slope of RS increases at this transition. As a result of these two factors, the value of RS in the MF is almost entirely due to the value of cellcell coupling resistance of the MF (e.g., see Equation 4, which separates the contributions of DF and MF to RS). The solid lines represent a best fit to the data using Equation 2 and Equation 3, which assume two values of the effective intracellular resistivity (Ri): RDF is the value of Ri in DF and RMF is the value in MF (
8 connexin +/+, +/-, and -/- mice. RDF is accurately determined from the first (outermost) measurement of RS. Note that the fits to +/+ and +/- data virtually overlay even though RDF is significantly higher in the +/- data (see Table 1). This is because RMF is about the same in these two classes of lenses, and as noted above, RMF dominates the value of RS as one records near the center of the lenses. If the effective intracellular resistivity is multiplied by the fiber cell width, w
3 µm, the reciprocal of that product is approximately the gap junctional coupling conductance per area of cellcell contact. For example, in lenses from +/+ mice, RDF = 3.5 K
-cm and 1/(RDF w) = 1.05 S/cm2.
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Table 1 summarizes the normalized coupling conductance based on the values of RDF and RMF in Fig 4, and on the average conductances from the 3 knockout mice studied in
In lenses +/- for either 3 or
8 connexin, there is about a 30% reduction in the coupling conductance of DF, and in lenses from the -/- mice the coupling conductance is reduced by 6070%. If each connexin contributed equally to the coupling conductance of DF, lenses from mice +/- for either connexin should have a 25% reduction in coupling conductance, and lenses from -/- mice should have a 50% reduction. However, in Table 1 the reductions are greater, as if each connexin contributed more than half the conductance. One should keep in mind that these numbers were derived from fitting the data in Fig 4. When generating data like those in Fig 4, it is difficult to accurately locate the position of the tip of the voltage recording microelectrode relative to spherical coordinates within the lens. This is a major source of scatter, which leads to uncertainty in the derived values of coupling conductance. Moreover, as just described, the lenses from -/- mice were obviously abnormal and there are likely to be indirect effects related to their poor health. With these limitations in mind, our interpretation of the data in Table 1 is that both connexins are functionally important in the DF and each connexin contributes about equally to the coupling conductance of DF.
Comparison of the conductances in the MF of the heterozygous lenses indicates that 3 +/- lenses have reduced coupling, whereas
8 +/- lenses have the same conductance as wild type. This suggests
8 connexin may not have a functional role in the coupling of MF. In lenses from
3 connexin -/- mice, the coupling conductance of MF is zero, which is also consistent with lack of functional
8 connexin in this domain. As described earlier, indirect effects in the lenses from connexin -/- mice are of concern. However, the abrupt reduction of coupling conductance to zero in the DF to MF transition cannot be simply attributed to an indirect effect. Moreover, because of the way the lens grows, the MF of these lenses were DF a week or two earlier, and at that stage, they were well coupled by junctions made of
8 connexin. Note that DF of lenses from
3 connexin -/- mice are well coupled by the standards of most cells, though the conductance is somewhat reduced from normal. Consideration of all these factors suggests that
8 connexin is not functional in the MF. However, inconsistent with this hypothesis, lenses from
8 -/- mice show a reduction in MF coupling conductance. Nevertheless, these lenses are grossly abnormal and there are several indirect effects, such as reduced lens size, increased input conductance, and depolarized resting voltage. Therefore, the reduced coupling may be an indirect effect of the
8 knockout. For example, intracellular Ca2+ homeostasis may be compromised, there may be abnormal protease activity, etc., leading to reductions in coupling conductance. The simplest interpretation of the changes in MF conductance is that the DF to MF transition results in a loss of functional
8 connexin, which in turn reduces the coupling conductance and leaves
3 as the only functional connexin.
Gap Junctional Gating
Cytoplasmic acidification has been shown to gate gap junctions closed in the DF region, but not in the MF region, of normal lenses from frog (8 connexin.
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We have determined the sensitivity of the gap junctions to cytoplasmic acidification in the DF and MF of 8 knockout lenses (Fig 5). Fig 5 A compares the effect of 100% CO2 on DF junctional resistance in +/+ and -/-
8 lenses. To facilitate this comparison, resistances are normalized to their initial values. In a 10-min exposure to 100% CO2, RP (Equation 1 and Equation 2) in lenses lacking
8 connexin increased by a factor of only 1.6, whereas RP in wild-type DF increased 4.7-fold. This suggests that homotypic
3 channels in the lens have a very limited ability to gate in response to acidification and that regulation of DF coupling requires the presence of
8 connexin. Indeed, the 1.6-fold increase in series resistance is very rapid and could be due to effects only on surface cell junctions, where
1 (Cx43) is the dominant connexin.
Fig 5 B shows the effect of 100% CO2 on the total coupling resistance of DF in series with a significant fraction of MF. Owing to spherical divergence of current flow, the resistance of the MF dominates, and the closure of channels in DF has a relatively smaller effect than that shown in Fig 5 A. Based on Equation 3 and a typical measurement where the +/+ lens radius a = 0.101 cm, the DF extends from radial location b to a, b = 0.8a, and the resistance is measured r centimeters from the lens center, r = 0.4a:
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(4) |
The resistance values in Equation 4 represent typical +/+ contributions of DF and MF to Rs. Our interpretation of the data from +/+ lenses is that CO2 has no effect on RMF, but causes the resistance of +/+ DF to go from 690 to
6,900
(i.e., RP varies from 3.5 to 35 K
-cm). Thus, the effect on total Rs is measurable but fractionally much smaller than that shown in Fig 5 A. In the lenses lacking
8 connexin, the resistance of DF increases by a factor of 1.6, according to Fig 5 A. This would represent about a 4% increase in total Rs of
8 -/- lenses (Fig 4) and would not be easily detectable, assuming RMF is not sensitive to CO2. Indeed, Fig 5 B shows no detectable increase in Rs for the
8 -/- lenses, providing direct evidence that homotypic
3 channels in MF are unable to gate in response to changes in pH, just as has been observed in other lenses (for review see
8 connexin -/- mice.
In 10 min, the effect of CO2 on the 8 -/- lenses is essentially complete, whereas homotypic
8 channels (in
3 -/- lenses;
3 and
8 channels continue to close for
30 min. Typical final values (after 30 min) of RP in two lenses from +/+ mice were 38 and 45 K
-cm. Curve fits of full impedance datasets (0.011,000 Hz) from lenses in normal Tyrode (no bubbling with CO2) of +/+ mice gave an average value of Re of 27 ± 17 K
-cm (mean ± SD, n = 11). If Re were the same after bubbling with 100% CO2, the value of RP should not have exceeded 27 K
-cm (i.e., Ri
in Equation 1, yields RP = Re). In 2 of the 11 fits, the values of Re exceeded 40 K
-cm, so it is possible that the CO2 data were from lenses with unusually high values of Re. However when lenses are exposed to CO2, their resting voltage depolarizes significantly (
3 -/- mice, the final values of RP were even larger (92 and 89 K
-cm). In these lenses, we were unable to obtain reasonable curve fits to impedance data using our equivalent circuit model of a normal lens, so the value of Re is not known. Thus, we cannot know for certain if the larger final values of RP have any significance with regard to closure of gap junction channels. Nevertheless we think it is unlikely, since any time the value of RP exceeds
27 K
-cm, all gap junction channels have probably closed. Thus we interpret these data as the total closure of all the homotypic
8 channels in DF junctions.
The data in Fig 5 suggest homotypic 3 channels are not closed by a drop in pH. If +/+ DF junctions comprise homotypic, independent
3 and
8 channels, which are parallel resistance paths, then the final resistance (following exposure to 100% CO2) between DF of +/+ lenses should not be greater than that recorded in the
8 -/- lenses, which have homotypic
3 channels. However, the final resistivity in lenses lacking the
8 connexin was 918 K
-cm, whereas that of +/+ lenses was 3845 K
-cm. Thus the presence of
8 connexin enables the total closure of all gap junction channels in DF of +/+ mice. These data suggest synergy between the
3 and
8 connexins, inasmuch as channels in junctional plaques containing mixed connexins gate closed more efficiently than would be expected for homotypic, independent channels. The simplest explanation for this observation is that all DF channels are heteromers, containing a mix of
3 and
8 subunits, with one
8 subunit being sufficient to cause closure of each channel. However, one must also consider indirect effects of loss of
8 connexin, such as changes in posttranslational processing of
3 connexin. Whatever the mechanism, this was an unexpected and unique observation that one connexin is regulated by the presence of another.
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DISCUSSION |
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The data presented here in connection with previous results (3 and
8 connexins have distinct roles in lens gap junctional communication:
3 connexin is necessary for the coupling of MF, whereas
8 connexin is necessary for the pH-sensitive gating of junctions coupling DF.
MF in lenses from mice lacking the 3 connexin also lack coupling. Lenses from
3 +/- mice have
50% less
3 connexin than wild-type lenses (based on quantitative Western blotting), and their MF have
50% less coupling conductance. Similarly, lenses from
8 +/- mice have
50% less
8 connexin than wild type, but in contrast, their MF have the same coupling conductance as wild-type lenses. These data suggest MF coupling conductance depends directly on the amount of
3 connexin and is independent of the amount of
8 connexin. This suggests that MF junctions do not contain functional
8 connexin. The one observation inconsistent with this hypothesis is the drop in MF coupling conductance in the lenses from
8 -/- mice. Many physiological properties of these lenses were abnormal, however, and it would be surprising if there were not some indirect effects on gap junctional conductance. Perhaps the more telling observation is that the MF are still well coupled in the
8 connexin knockout lenses.
Both connexins contribute about equally to the coupling conductance of DF. However, in the absence of 8 connexin, the lens homotypic
3 channels are unable to respond to a change in pH, whereas in the presence of
8 connexin, all DF junctional channels gate closed in response to acidification. Thus, the presence of
8 connexin may regulate the conductance contributed by
3 connexin.
The simplest explanation for the synergistic gating between the 3 and
8 connexins would be heteromeric channels (
3 and
8 connexins and if only one
8 subunit were sufficient to cause closure, then the gating data presented here would be explained. However, the simplest model to explain the MF conductance changes shown in Table 1 is that MF communication is via homotypic
3 channels. But if the channels are homomers in the MF, one would expect that they started out as homomers in the DF, which is inconsistent with the simplest explanation of DF gating. Therefore, a more complex model is necessary to explain both observations. A number of increasingly complex hypotheses can be formulated, but the available data do not distinguish the possibilities. Higher resolution biochemical and biophysical characterization of channels in both DF and MF is needed to provide a basis for a hypothesis on molecular mechanism.
In all species studied, the DF to MF transition results in a loss of gap junctional gating in response to acidification (for review see 8 connexin (
3 connexin is not pH sensitive, even in DF where this connexin is thought to be intact, at least in wild-type lenses. Thus, regardless of the connexin structure or composition of lens gap junction channels, these data imply coupling of MF would be insensitive to pH. Therefore, the more perplexing issue is the mechanism of pH gating of DF junctions. Interestingly, when exogenously expressed in oocytes, homotypic
3 junctions are sensitive to pH (
3 junctions in the DF of
8 knockout mice. This difference in the properties of endogenous and exogenously expressed
3 connexin may reflect the presence of different regulatory proteins, or it may be due to differences in posttranslational processing. Understanding this difference may lead to a better understanding of gap junctional gating in DF of the lens.
In the INTRODUCTION, we suggested two major roles for lens gap junctions: (1) setting the resting voltage of inner fiber cells and, thus, maintaining volume regulation in these cells; and (2) conducting the lens circulation to the inner fiber cells and, thus, providing delivery of glucose for metabolism and homeostasis. In lenses from 3 -/- mice, MF were uncoupled from the surface cells. These fibers had a depolarized resting voltage that is consistent with hypothesis 1. Moreover, the central MF had a complete breakdown in homeostasis and developed a dense cataract that is consistent with hypothesis 2. These observations, of course, do not prove the hypotheses, but once again the data fail to contradict them. Surprisingly, it appears that these roles depend on the presence of
3 connexin and are lost when
8 is the only fiber cell connexin. Thus, the expression of
3 connexin in fiber cells is apparently the lens resolution to the problem of maintaining gap junctional communication throughout the life of fiber cells where connexin turnover is not possible. Lenses from
8 -/- mice had reduced gap junctional coupling, but MF remained in communication with the surface cells. Although these lenses were clearly compromised, they did not develop a dense central cataract, which is consistent with the presence of a reduced circulation. The major difference between coupling of fiber cells in +/+ versus -/-
8 knockout lenses was the loss of pH sensitivity in DF junctions. Yet, the
8 -/- lenses display many secondary changes, such as reduced size, altered membrane transport, depolarized resting voltage, and rapid deterioration after dissection. It is possible that all of these secondary effects are a consequence of loss of gating. The lack of
8 connexin is thought to disrupt early development (
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Footnotes |
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* Abbreviations used in this paper: DF, differentiating fiber; MF, mature fiber.
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Acknowledgements |
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We would like to dedicate this paper to Dr. Bernie Gilula, who passed away on September 27, 2000. To those of us at Scripps, Bernie was a mentor, close colleague, and friend. To those of us at SUNY, he was a valued collaborator and friend. We shall all miss him.
This work was supported by the National Institutes of Health grants EY06391 (to R.T. Mathias), EY11093 (to N.M. Kumar), EY12142 (to N.B. Gilula and N.M. Kumar) and GM37904 (to N.B. Gilula).
Submitted: 9 July 2001
Revised: 17 September 2001
Accepted: 18 September 2001
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