Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

*Electrically silent channels

in the lens. Thus, lenses in which the inwardly rectifying K+ channels dominate exhibit a higher potential than those in which the outwardly rectifying channels predominate. The Maxi-K channel has been identified in all species of lenses investigated to date, with the possible exception of the frog lens. The biophysical properties of the Maxi-K channel are such that it is inactive at normal resting membrane potentials. Activation of this type of channel can occur upon elevation of cytoplasmic Ca2+. Thus, the recent finding (see below) that the lens contains an array of membrane receptors capable of mobilizing intracellular Ca2+ suggests that Maxi-K channels could play a role in the regulation of lens membrane potential, a notion that warrants further investigation.

A role for K+ channels in fiber cells has not been as extensively investigated as in epithelial cells. It appears that isolated differentiating fiber cells are still capable of maintaining a negative membrane potential (Donaldson et al., 1995) and exhibit conductances that resemble the potassium conductances observed in epithelial cells (Fig. 6.3a). However, as the fiber cells differentiate and elongate, they become depolarized and their membrane properties become dominated by a large nonselective leak conductance (Fig. 6.3b). Thus, fiber cell differentiation appears to be associated with a loss of potassium conductance (Fig. 6.3c).

6.3.2. Sodium Conductance

Patch clamp experiments on epithelial cells have shown that, in addition to a K+ conductance, epithelial cells often contain a Na+ leak conductance, which may be due to a stretchactivated cation channel in amphibian lenses (Cooper et al., 1986) or a tetrodotoxin-sensitive Na+ channel (Watsky et al., 1991). The relevance of these channels to lens physiology remains uncertain. While it is believed that inner fiber cells contain a Na+ or cation conductance, the molecular nature of the channels that sustain this conductance is not presently known. Interestingly, in whole lens experiments, removal of extracellular Ca2+ induces an increase in the fiber cell cation conductance (Rae et al., 1992). Membrane currents of isolated elongated fiber cells bathed in zero Ca2+ to prevent fiber cell globulization also exhibit a dominant cation conductance (Eckert et al., 1998). One of these currents (Fig. 6.3b) exhibited a striking similarity to the so-called hemichannel currents recorded from single

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Figure 6.3. Conductance properties of acutely isolated cells from the rat lens. (a) Representative current trace recorded from a short ( 60 µm) fiber cell bathed in normal Ringer’s. (b) A large nonselective hemichannel-like conductance recorded from a long ( 180 µm) fiber cell bathed in Ca2+-free Ringer’s. (c) A plot of specific conductivity (membrane conductance per area of membrane) versus cell length for isolated epithelial and fiber cells. This plot shows that, in normal Ringer’s, the longer the cell, the lower the membrane conductance. However, incubation of longer fiber cells in Ca2+-free Ringer’s appears to induce a large increase in the specific conductivity, presumably via the activation of the hemichannel-like conductance.

oocytes expressing the gap junction protein connexin 46 (Ebihara and Steiner, 1993; Ebihara et al., 1995). In either cell system, these currents were only activated when Ca2+ was reduced and the cells were depolarized. Hence, hemichannels are unlikely to open in the lens under normal conditions, a prediction also consistent with electrical measurements of whole lenses, which characterize the fiber cells as electrically “tight” (Mathias et al., 1997). However, because lens depolarization is often observed early in cataractogenesis, it is conceivable that hemichannel currents become activated under pathologic conditions. This would lead to further depolarization of the fiber cells, causing an influx of ions and water and, ultimately, the cell swelling and globulization that is typically seen in cortical cataractogenesis (Bond et al., 1996).

6.3.3. Chloride Conductance

Patch clamp experiments have to date failed to identify a Cl− selective current in lens epithelial cells (Mathias et al., 1997; Eckert, unpublished observations). More recently, transcripts for the Cl− channel isoforms ClC2 and ClC3 where extracted from a cDNA library prepared from human lens epithelium (Shepard and Rae, 1998). In other expression systems, the

activity of these Cl− channel isoforms is increased by exposure to hypotonic solutions. Hence, in lens epithelial cells, these channels may be inactive under normal conditions and become activated only following changes in cell volume. Measurement of lens potential following anion substitution indicates that the fiber cells have a significant Cl− conductance (Mathias et al., 1985). Consistent with this notion, Cl− channels have been identified in membrane vesicles derived from bovine lens fiber cells (Zhang and Jacob 1994) and in isolated differentiating fiber cells (Zhang et al., 1994; Zhang and Jacob, 1996). In both preparations, Cl− currents were blocked by a wide variety of Cl− channel blockers, including tamoxifen and NPPB. Tamoxifen is a highly potent and selective blocker of p-glycoprotein, the multifunctional protein product of the multidrug resistance gene (MDR1). P-glycoprotein is also considered to be a regulator of volume-activated Cl− channels. Interestingly, the addition of either tamoxifen or NPPB to organ-cultured lenses inhibits the ability of the lenses to volume regulate and causes lens opacification (Zhang et al., 1994; Zhang and Jacob, 1996; Tunstall et al., 1999). A role for Cl− ions in lens volume regulation is more fully discussed later.

6.4. Lens Cells Are Connected by Gap Junction Channels

It has been long recognized that cells throughout the lens are extensively coupled via gap junctions (Goodenough, 1979; Goodenough et al., 1980; Mathias et al., 1981; Lo and Harding, 1986). Gap junctions connecting the fiber cells are particularly abundant and have been observed predominantly on the membrane broadsides (see chap. 4; see also Gruijters et al., 1987a, 1987b ). This arrangement facilitates the convection of ions, solutes, and water in a radial direction, consistent with the outflow pathway of the lens circulation model. The first membrane protein correctly identified as a cell-cell channel-forming polypeptide in the lens is MP70, now known as connexin 50 (Kistler et al., 1985; Kistler et al., 1988; White et al., 1992). A closely related protein, connexin 46, was also identified, and together they form the gap junctions connecting fiber cells (Paul et al., 1991). The two isoforms assemble with each other to form heteromeric connexons (Jiang and Goodenough, 1996). A third isoform, connexin 43, was identified as the predominant gap junction protein in the lens epithelial cell membranes (Beyer et al., 1989). Other species isoforms were cloned from the lens as follows: chicken connexin 56 (Rup et al., 1993) and bovine connexin 44 (Gupta et al., 1994a) are homologous to rat connexin 46 (Paul et al., 1991); chicken connexin 45.6 (Jiang et al., 1994), ovine connexin 49 (Yang and Louis, 1996), and human connexin 50 (Church et al., 1995) correspond to mouse connexin 50 (White et al., 1992). No novel connexins have been found in the lens over and above the three isoforms initially identified.

Lens fiber cell connexins were isolated and confirmed to form channel structures by negative stain electron microscopy. This was possible because connexin 50 and 46 are soluble in mild detergents that leave other proteins embedded in the membrane (Kistler and Bullivant, 1988). Reconstitution of the solubilized channel structures with exogenous lipids resulted in the reassembly of gap junctions that were reminiscent of their native counterparts in lens fiber cell tissue (Kistler et al., 1994).

Lens connexins were expressed in Xenopus oocyte pairs to verify that they individually had the ability to form communicating channels (White et al., 1994). All three connexins formed channels that were sensitive to voltage and acidification. Patch clamping of acutely isolated epithelial cell pairs (Donaldson et al., 1994) and fiber cell pairs (Donaldson et al., 1995) established that channels with similar electrical gating properties indeed exist in the

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lens. Notably, the differential expression of connexins in epithelial and fiber cells results in significant changes in voltage dependence and unitary conductance, suggesting that altered gap junction permeabilities play a role in lens development. Whole lens experiments are also consistent, in that they were able to demonstrate pH-mediated uncoupling of fiber cells electrophysiologically (Baldo and Mathias, 1992; Emptage et al., 1992) and by dye transfer (Eckert et al., 1999) in the intact cortical tissue.

Phosphorylation of connexins appears to further regulate gap junction permeability in the lens. In lens cell cultures, activation of protein kinase C with a phorbol ester phoshorylated connexin 43 and significantly decreased dye transfer between epithelial cells (Reynhout et al., 1992). The situation is less clear for fiber cells. Coupling between fiber cells of cultured ovine lentoids expressing connexin 46 and 49 was unaffected by the phorbol ester treatment (Tenbroek et al., 1997). In contrast, the same treatment significantly reduced dye transfer between fiber cells in cultured chicken lentoids (Berthoud et al., 2000). It was independently confirmed that the fiber cell connexins were also phosphorylated in the whole lens and that protein kinase C was indeed involved (Jiang and Goodenough, 1998). Thus it appears possible that gap junction permeability is differentially regulated at different stages of lens development not only by a switch in connexin expression but also by protein kinase C–dependent connexin phosphorylation. However, there may be species variations with regard to the latter.

Lens casein kinase was also found to phosphorylate the connexin 50 homologs in chick and sheep (Cheng and Louis, 1999; Yin et al., 2000). The function of this phosphorylation event appears to be the destabilization and degradation of the connexin. Ser363 of connexin 45.6 was phosphorylated. In a mutant that had the serine replaced with alanine, this phosphorylation did not occur, and the result was a longer half-life than possessed by the wild-type connexin (Yin et al., 2000). Connexin 56 also seems to be affected in a similar way: One study detected two pools of connexin 56 that were phosphorylated but had different molecular weights (Berthoud et al., 1999). One form had a half-life of only a few hours, the other a half-life of days. These findings strongly suggest that gap junction stability in the lens is regulated by phosphorylation.

As another form of posttranslational processing, connexin 46 and 50 were both found to be cleaved upon fiber cell maturation at about the time when the cell nuclei were degraded (Fig. 6.4a; Kistler and Bullivant, 1987; Lin et al., 1997). This cleavage, accomplished by lens endogenous calpain, removes a significant portion of the cytoplasmic carboxyl tail and leaves the channel-forming portion in the membrane (Kistler et al., 1990; Lin et al., 1997). In the case of connexin 50, it has been possible to determine precisely the cleavage site in the molecule, allowing the construction of an appropriate truncation mutant for expression in Xenopus oocyte pairs (Fig. 6.4b). The functional consequences of the cleavage could thus be established (Fig. 6.4c), including the abolishment of the pH gating of the cell-cell channels (Lin et al., 1998). Similar efforts with connexin 46 have failed so far – the precise cleavage site remains unknown. However, it is reasonable to assume that its cleavage would have a similar effect on channel gating, as the same phenomenon was observed among other connexins. (Delmar et al., 2000).

The functional implications of this cleavage could be crucial for the lens. The lens nucleus is mildly acidic and has a pH around 6.5 (Mathias et al., 1991; Mathias et al., 1999), which is sufficient to close gap junction channels and thus uncouple the core region from the peripheral lens tissue. Thus, the lens has developed an elegant mechanism to keep the deeper-lying fiber cells communicating by simply abolishing the pH gating of the cell-cell channels through truncation of the connexins. That cell-to-cell communication in the lens

Figure 6.4. (See color plate IX.) Cleavage of connexins in the lens. (a) Top panel: axial section through the bow region of a mouse lens showing the coincidental loss of nuclei (red) and the carboxyl tail of connexin 50 (green). Bottom panel: equatorial section doublelabeled with two different connexin 50 antibodies, one specific for the carboxyl tail (green) and the other specific for the cytoplasmic loop (red). This demonstrates that the membraneembedded channel-forming portion of connexin 50 persists toward the center of the lens. Scale bar: 100 µm. (b) The Ca2+-activated protease calpain cleaves the cytoplasmic tail of connexin 50 at amino acid residues 290 and 300. (c) Truncated recombinant connexin 50, which mimics the in vivo cleaved form, makes functional gap junction channels in Xenopus oocytes. Upon acidification of the oocytes by exposure of the bath solution to 100% CO2, it becomes evident that the wild-type connexin 50 is pH-sensitive while the truncated version has lost pH-gating.

nucleus indeed occurs and is pH insensitive was verified in electrophysiological (Baldo and Mathias, 1992) and dye transfer experiments (Eckert et al., 2000).

The production of knockout mice for all three connexins expressed in the lens means that it is now possible to determine the contribution each isoform makes to lens transparency. In each case, the disruption of an individual connexin gene results in cataractogenesis. In the case of connexin 43, which is the predominant isoform forming the cell-cell channels in the epithelium (Beyer et al., 1989; Donaldson et al., 1994), the normally close cellular apposition is severely disrupted (Gao and Spray, 1998). Specifically, fiber cells are separated from the apical surfaces of the epithelial cells, and large vacuolar spaces are apparent between the fiber cells, most prominently in the deeper lens cortex. This damage phenotype is similar to those observed in cortical osmotic cataracts.

In contrast to disruption of the connexin 43 gene, disruption of the connexin 46 gene results in nuclear cataract (Gong et al., 1997). A detailed electrophysiological analysis of

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these lenses showed that the differentiating fiber cells remain coupled, albeit at reduced levels, consistent with the apparently normal distribution of connexin 50 in these cells (Gong et al., 1998). Surprisingly, however, in mature fiber cells deeper in the lens, the coupling conductance approached zero, despite the fact that connexin 50 is present in its cleaved form, which still produces functional channels in the Xenopus oocyte system (Lin et al., 1998). The loss of communication deeper in the lens correlates with the formation of nuclear cataract, indicating strongly that the proper functioning of gap junction channels is essential for the maintenance of homeostasis in the lens core region. It further indicates that the cleaved channels in the lens nucleus might require both fiber cell connexin isoforms to be functional (unlike the oocyte model system).

The disruption of the connexin 50 gene affects not only the lens but also eye development (White et al., 1998). The null mutant mice exhibit microphthalmia and nuclear cataracts. Lens mass is reduced 46% from normal, and opacities become evident shortly after birth. Analysis by microinjection of fluorescent tracers indicate that coupling between all cell types persists in all regions of the knockout lenses. The results suggest that both normal eye growth and maintenance of lens transparency depend on the unique properties of connexin 46 and 50 in combination.

Dysfunctions of connexin 46 or 50 have also been associated with certain forms of inherited congenital cataract in humans and mice (Mackay et al., 1997; Mackay et al., 1999; Shiels et al., 1998; Steele et al., 1998). The human cataracts are of the zonular pulverent (nuclear) type, similar to those observed in the knockout mice. A connexin 50 missense mutation (P88S) was further characterized in Xenopus oocyte pairs and shown to act in a dominant negative manner when coexpressed with normal human connexin 50 (Pal et al., 1999). One P88S mutant subunit per gap junction channel was sufficient to abolish channel function. These observations underline the importance of intercellular communication to the generation of the lens internal circulation.

6.5. Na+ Pump Activity Is Greatest at the Lens Equator

The active extrusion of Na+ by the Na+ pump is critical to the generation of the circulating ion fluxes that drive the lens internal circulation. The circulation model predicts that most Na+/K+-ATPase activity is concentrated in the equatorial region of the lens. Gao et al. (2000) also measured the pump density and estimated that the total Na+/K+ pump activity per unit area of lens surface was about 20 times larger at the equator than at the anterior pole. This is consistent with an earlier study, in which the researchers separated the capsule, with the epithelium attached, from the bulk of the lens and then dissected a superficial anteriorequatorial cortex segment and a superficial posterior cortex segment (Alvarez et al., 1985). The principal Na+/K+-ATPase activity was found in the superficial anterior-equatorial cortex segment. By comparison, enzyme activity was 1.6 times less in the capsule epithelium and negligible in the posterior cortical segment. In older fiber cells deeper in the lens, pump protein was retained but had lost its functional ability (Delamere and Dean, 1993).

In addition to an increased activity of the Na+/K+-ATPase at the equator relative to the poles, a number of investigators have shown that there is also a change in the molecular composition of the Na+ pump. There are at least three major isoforms of the Na+/K+- ATPase α subunit, and all three have been identified in the lens (Moseley et al., 1996; Garner and Kong, 1999; Tao et al., 1999). All three studies agree that these isoforms are differentially expressed but are inconsistent regarding the precise localization: In the first

study, which used rat lenses, the epithelium contained predominantly α1 and α2 and less α3, while fiber cell membranes contained only α1 (Moseley et al., 1996). In the second study, which used bovine and human lenses, α1 and α3 were localized in the central epithelium, and α2 and α3 were found in cortical fiber cells (Garner and Kong, 1999). In the third study, abundant α2 and α3 were found in the epithelium of rabbit lenses, and minor amounts of α3 were detected in fiber cells form (Tao et al., 1999). Finally, in a recent report, α2 was the predominant isoform at the anterior pole of frog lenses, and α1 was predominant at the equator (Gao et al., 2000). While these inconsistencies may simply reflect species differences, the physiological relevance of the observed differential expression patterns is intriguing, especially in the light of recent reports that indicate that regulation of the Na+ pump is isoform specific. Thus, the localization of a specific isoform at the equator may confer the ability to regulate lens circulation.

6.6. Water Flow across Lens Cell Membranes Is Enhanced by Aquaporins

The lens circulation model postulates that the circulating ion fluxes are accompanied by water flow. This necessitates that both epithelial and fiber cells have a significant water permeability. Consistent with this idea is the finding that each cell type expresses a distinct type of water channel or aquaporin (AQP) isoform. In the epithelial cell membranes, water permeability is enhanced by AQP1, while in the fiber cells, AQP0 (orginally called the main intrinsic polypeptide [MIP]) is responsible (Stamer et al., 1994; Patil et al., 1997; Lee et al., 1998; Hamann et al., 1998). When expressed in Xenopus oocytes, both aquaporin isoforms significantly increased the water permeability of the oocyte membrane, but AQP1 was about 40 times more effective as a water channel than AQP0/MIP (Chandy et al., 1997). In accordance with this, the water permeability of lens epithelial membranes is much greater than that of the fiber cell membranes (Fischbarg et al., 1999; Varadaraj et al., 1999). This difference may be crucial for the proper functioning of the lens circulation system, as it allows the epithelial membranes to cope with the large water flow arising from the cumulative entry into the large number of fiber cells.

MIP was not always recognized as a water channel and in fact has been attributed several functions over time. Initially isolated (Broekhuyse et al., 1976) and cloned (Gorin et al., 1984) from bovine lenses, MIP is the most prominent of all fiber cell membrane proteins and in some species constitutes up to 60% of total membrane protein. It is present throughout the lens cortex and nucleus and was first localized in the 11to 13-nm “thin” fiber membrane junctions, which were for some time wrongly interpreted as communicating junctions (Bok et al., 1982; Sas et al., 1985). This mistake was recognized when it was demonstrated that MIP was unable to form communicating channels in the Xenopus paired oocyte expression system (Swenson et al., 1989). Later, MIP was demonstrated to form relatively large nonselective ion channels in planar lipid bilayers (Ehring et al., 1990; Shen et al., 1991; Modesto et al., 1996), a capability that could not be reproduced by expressing MIP in oocytes. Only more recently has MIP been recognized as the founding member of the MIP family of water and solute channels (Park and Saier, 1996; Heymann and Engel, 1999). When expressed in Xenopus oocytes, MIP increased membrane permeability for water and glycerol (Kushmerick et al., 1995; Mulders et al., 1995; Chandy et al., 1997). This water channel activity was confirmed also for the lens (Fig. 6.5a) on the basis that fiber cell membranes containing wild-type MIP had a significantly larger water

162 Joerg Kistler, Reiner Eckert, and Paul Donaldson

Figure 6.5. Water channels in the lens. (a) The water permeability (PH2 O ) of epithelial cells that express AQP1 exceeds that of membrane vesicles prepared from fiber cells containing MIP (Varadaraj et al., 1999). (b) A common structure for lens water channels. Projection maps for AQP1 and MIP show that these two membrane proteins have a similar structure (Hasler et al., 1998). The full width of the image corresponds to 6.4 nm.

permeability than fiber cell membranes containing a mutant form of MIP (Varadaraj et al., 1999).

MIP is unique among the aquaporins in that its water channel activity is pH dependent. When expressed in Xenopus oocytes, reduction of the pH to 6.5 increased the water transport activity of MIP 3.4 times (Nemeth-Cahalan and Hall, 2000). The prevailing pH in the lens nucleus is similar to this value due to the accumulation of lactate as the end product of glycolysis (Bassnett et al., 1987; Mathias et al., 1991; Mathias et al., 1999). Hence, it is possible that the water transport activity of MIP in the lens core is enhanced by the lower pH, which could be a mechanism facilitating the penetration of the circulating ion and water fluxes deeper into the lens. This feature of MIP could even represent a feedback mechanism whereby a decrease in intracellular pH could act as a signal of increased metabolic activity and thus could be a stimulus for greater fluid flow in regions of the lens with elevated metabolic activity.

Further support for MIP’s role as a water channel is provided by its structural homology to the archetypical water channel AQP1. Following solubilization from fiber cell membranes with detergent, MIP was isolated as tetramers (Aerts et al., 1990; Konig et al., 1997; Hasler

et al., 1998), which upon reconstitution with lipids crystallized into highly ordered square arrays (Hasler et al., 1998). Projection maps initially at 0.9 nm (Hasler et al., 1998) and then at 0.6 nm (Fotiadis et al., 2000) revealed several protein densities, which could be attributed to transmembrane helices, and displayed a configuration around a central pore similar to that observed for AQP1 (Fig. 6.5b). The agreement between structural and functional data makes a strong case that MIP indeed plays a crucial role in facilitating the flow of water that follows the circulating ion flux in the lens.

Finally, and somewhat ironically, an interesting aspect of the two-dimensional crystals of MIP is that most turned out to consist of two membrane sheets that faced each other with their extracellular sides. They were held together by tightly fitting tongue-and-groove interactions between the MIP molecules in the apposing membranes (Fotiadis et al., 2000). This feature of the in vitro reconstituted membranes is consistent with all earlier reports, which identified MIP as a junction-forming protein in the lens. Thus it is likely that MIP has dual functions: first as a water channel, second as a cell-to-cell adhesion protein. Consistent with this, MIP is present in junctional as well as nonjunctional portions of the fiber cell membranes (Fitzgerald et al., 1983; Paul and Goodenough, 1983). It appears possible that MIP directly contributes to the unusually tight packing of the fiber cells that is so important for lens transparency. Such a key role is supported by the observation that mutations of MIP cause cataracts in mice (Shiels and Bassnett, 1996).

6.7. Specialized Transporters Serve Nutrient Uptake

The circulating current creates a net flux of solute that generates an extracellular fluid flow that in turn convects nutrients toward the deeper-lying fiber cells, while the intracellular flow removes waste products from the intracellular compartment. For this model to be correct, the fiber cells need a mechanism for importing the nutrients that are being convected to them. Glucose is the principal fuel that the lens uses to support growth and homeostasis. In the lens, the epithelium and differentiating fiber cells are capable of oxidative phosphorylation, but the mature fiber cells, having lost their mitochondria, must rely solely on glycolysis for energy production. The lens is bathed by the aqueous humor, which contains glucose levels that mirror those in the plasma. Hence, lens cells near the periphery have access to an abundant supply of glucose; however, the supply of glucose to the deeper-lying fiber cells is likely to be limited by the tortuous nature of the extracellular space.

Transport studies to identify the site of glucose uptake in the lens are not entirely consistent with each other. One report identified the epithelium as the predominant site of glucose transport (Goodenough et al., 1980). Another study demonstrated that, in addition to the epithelium, fiber cells also have the ability to transport glucose. This was concluded from the observation that the capacity of glucose transport was comparable at both the anterior and posterior lens surfaces (Kern and Ho, 1973). Furthermore, approximately half of the total lens glucose transport remained following removal of the capsule and the adhering epithelial cell layer (Giles and Harris, 1959). In yet another study, glucose transporters were found predominantly in the lens nucleus and at lesser levels in the cortex (Lucas and Zigler, 1987, 1988; Kaulen et al., 1991). However, this localization was based on the binding of cytochalasin B to the transporters, and concerns remain about the specificity of the reagent. Also, the result is inconsistent with the observation that enzyme activities involved in the breakdown of glucose are greatest in the cortex and decrease toward the center of the lens (Zhang and Augusteyn, 1995). It appears that both the epithelial and cortical fiber cells are capable of glucose uptake.

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Figure 6.6. (See color plate X.) Differential expression of glucose transporters in the lens. An axial section of a rat lens showing the expression of GLUT1 (red) on the basolateral aspect of the epithelium and GLUT3 (green) in the differentiating fiber cells. Scale bar: 25 µm.

In other tissues, glucose uptake from the extracellular fluid is mediated by members of the glucose-facilitated transporter (GLUT) family (Gould and Holman, 1993). The identification and localization of glucose transporter isoforms in the lens has been controversial. For example, one study localized the glucose transporter GLUT1 in cortical fiber cells (Mantych et al., 1993), but another study reported a negative result for the same isoform (Kumagai et al., 1994). GLUT2, GLUT3, and GLUT4 were also investigated but could not be detected. A more recent study reported an absence of GLUT2 and GLUT4 in the rat lens but found strong evidence for the expression of GLUT1 and GLUT3 (MerrimanSmith et al., 1999). The latter two isoforms were reliably identified and localized at both transcript and protein levels. GLUT1 is predominantly expressed in the rat lens epithelium and differentiating cells at the equator, whereas GLUT3 expression is strongest in cortical fiber cells (Fig. 6.6). This differential expression is consistent with the different glucose environments these cells are exposed to. GLUT1 has a Km appropriate for the abundant glucose supplies in the aqueous humor. GLUT3 has a lower Km than GLUT1 and works most effectively in situations where glucose supplies are more restrictive, as is likely to be the case for deeper-lying fiber cells.