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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Physiology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
Submitted 22 July 2007 ; accepted in final form 20 March 2008
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ABSTRACT |
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electrophysiology; potassium channel; anion channel; nonselective cation channel
To date, efforts to confirm these spatial differences in membrane permeability have focused on lens epithelial cells since these surface cells are easy to obtain and maintain in culture and are therefore amenable to study using the patch-clamp technique. In isolated rodent lens epithelial cells, a Ba2+-sensitive inwardly rectifying K+ (Kir) channel has been shown to be active (43, 49), and the application of Ba2+ to these cells caused a depolarization of their membrane potential toward 0 mV (9). However, the application of Ba2+ to the intact rodent lens only causes a 10- to 20-mV depolarization (11, 48), indicating a nonexclusive role for Kir channels in setting overall lens potential. This suggests that either epithelial or fiber cells contain additional conductances that contribute to the resting potential of the intact lens (37). Consistent with this idea, a variety of other K+ conductances have been identified by patch-clamp studies conducted in the lens epithelium; however, not all of these channels are found in every species or even in any one cell (9). A delayed outwardly rectifying K+ conductance that is sensitive to tetraethylammonium (TEA) has been found in the lens epithelium of every species studied (for a review, see Ref. 37), but it is largely closed at potentials in the physiological voltage range, contributing only a fraction of its peak "active" conductance in cells at rest (43). TEA is only a weak depolarizer of whole lenses (34), a result consistent with the view that these channels play a minimal role in setting lens resting membrane potential. Ca2+-activated K+ (KCa) currents are ubiquitous in lens epithelia and are blocked by both Ba2+and TEA (46, 50). KCa channels are normally closed at physiological intracellular Ca2+ concentrations but are open if levels rise into the micromolar range, with their open probability increasing further with membrane depolarization. Once again, this strong Ca2+ dependence indicates that KCa channels probably do not play a major role in the maintenance of the lens potential. Thus, despite considerable characterization of the permeability properties of the lens epithelium, no one conductance appears to set the lens membrane potential (Vm), which suggests that the fiber cells themselves may contribute significantly to the lens potential.
Unfortunately, very little is known at the cellular level about the membrane properties of fiber cells. These elongated, highly differentiated cells are notoriously difficult to isolate and maintain in solutions that are amenable to electrophysiological analysis of their membrane properties, and past efforts to produce viable isolated fiber cell preparations have been only partially successful (13, 18, 19). Isolated lens cell preparations tended to contain only very short, newly differentiated fiber cells that exhibited an epithelial-like K+ conductance (13). Unfortunately, the membrane properties of isolated fiber cells degraded over time by activation of leak currents, often in midrecording, (13, 18, 41). In a single study, Jacob and co-workers (56) obtained short viable fiber cells from the bovine lens by enzymatic dispersion and plated them in physiological media embedded in the surface of low-temperature agar; however, difficulties in maintaining membrane integrity under whole cell patch clamp in the presence of Ca2+ makes such experimentation problematic. Bhatnagar and co-workers (6) obtained a preparation of highly elongated fiber cells from the rat lens by exposure to a nonionic trypsin solution that was heavily buffered to remove extracellular Ca2+. In this preparation, reintroduction of Ca2+ induced vesiculation, and this process could be significantly delayed by a variety of nonselective cation and Ca2+ channel blockers, suggesting that Ca2+ influx is a key trigger of vesiculation. Based on these observations, Eckert et al. (18, 19) obtained a preparation of isolated fiber cells that were amenable for patch-clamp analysis by the use of physiological Ringer solutions that lacked Ca2+. Unfortunately, while removal of extracellular Ca2+ prevented vesiculation, subsequent electrophysiological analysis of these cells indicated that Ca2+ removal resulted in the activation of a large membrane leak that appeared to resemble currents mediated by Cx46 hemichannels expressed in oocytes (17, 19). A similar increase in membrane leak was observed in whole lenses upon the removal of extracellular Ca2+, which could be blocked by the addition of a variety of multivalent cations (48).
Taken together, the above studies suggest that while blockade of Ca2+ influx is key to preventing fiber cell vesiculation, this needs to occur in the presence of extracellular Ca2+ if the activation of leak conductances are to be avoided and physiologically relevant membrane properties recorded from isolated fiber cells. Recently, we have shown the inclusion of the multivalent nonselective cation channel inhibitor Gd3+ in Ca2+-containing Ringer solutions enables physiologically relevant membrane currents to be recorded from isolated elongated (>120 µm) lens fiber cells (54). In this study, we present our analysis of a larger data set of whole cell recordings from isolated fiber cells that cover a wider range of cell lengths and therefore different stages of fiber cell differentiation. Our analysis indicates that the membrane properties of fiber cells undergo differentiation-dependent changes as fiber cells elongate and become internalized. The differentiation of epithelial cells into elongated fiber cells is accompanied by a switch from dominant epithelial-like K+ conductances in short fiber cells to a more dominant contribution from Cl– and Na+ conductances in longer, more differentiated fiber cells. These data represent the first confirmation at the cellular level of spatial variations in ion permeabilities within the lens cortex, a key postulate of the lens internal circulation model (35, 37).
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METHODS |
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20°C).
Isolation of lens fiber cells.
Three- to four-week-old Wistar rats were killed by CO2 asphyxiation and spinal dislocation in accordance with protocols approved by the University of Auckland Animal Ethics Committee. Enucleated eyes were placed into AAH on the stage of a dissecting microscope. Four radial cuts were made from the optic nerve head through the sclera as far as the border of the cornea. Two of the scleral flaps were grasped with forceps and everted to free the lens. The lens was then dissected from the ciliary body by cutting the suspensory ligaments. Sharpened forceps were then used to gently remove the capsule and epithelial and fiber cells attached to it. Capsules with adherent fiber cells were transferred immediately to an Eppendorf tube filled with 1 ml of collagenase dissociation buffer in a water bath at 35°C for 35–40 min. Fiber cells were lightly vortexed and centrifuged at 1,000 rpm for 2 min before gentle resuspension in a minimal volume of AAH (200 µl) containing 2.5 mM Ca2+ with either 1 or 3 mM Gd3+. Isolated cells were plated into a recording chamber, the bottom of which was formed by a poly-L-lysine-coated glass coverslip. After cells were left to settle and adhere for 5 min, the bath was overlaid with solution to give a bath volume of 1 ml.
Electrophysiological recording.
The chamber was mounted on the stage of an inverted microscope (Nikon Eclipse, Nikon, Melville, NY) fitted with differential interference contrast optics. The recording chamber was constantly perfused under gravity at a rate of 1–2 ml/min. Test solutions were injected in 1.5-ml volumes via a custom-made low-deadspace manifold fabricated from 10-µl pipette tips using a TTL-driven peristaltic pump (Gilson Minipulse, Middleton, WI) and a network of multiway taps to control solution flow. Wash out of solutions was achieved via continuous aspiration of the bath surface from a connected bath compartment to give a constant bath volume. All single cell electrophysiological experiments were conducted at "room temperature" (20°C) and used solutions that contained 1 mM GdCl3 to prevent fiber cell vesiculation. Patch clamping was performed using a Multiclamp 700A amplifier (Axon Instruments, Union City, CA) that was fitted with two matching high-current headstages. Patch electrodes were mounted directly on these headstages, which were positioned using a pair of piezoelectric manipulators (Burleigh PCS-5000, Exfo Life Sciences, Missisuaga, Ontario, Canada). Because of their elongated morphology, there was an initial concern that voltage clamp in the distal parts of the fiber cell could be partially attenuated, leading to space clamp errors that may affect the accuracy of calculated membrane parameters (26, 28). In an effort to minimize such errors, seals were formed as close to the midpoint of a fiber cell as practicable, thereby halving the linear distance required to voltage clamp cell extremities, and series resistance (Rs) was kept to a minimum (17.21 ± 0.76 M
, n = 196). Data were digitized using a Digidata 1322A SCSI analog-to-digital converter (Axon Instruments) into a PC Pentium II 266-MHz computer running the pCLAMP version 8.1 software package (Axon Instruments). Images were acquired via a cooled charge-coupled device camera (Photometrics Imagepoint, Roper Scientific, Tuscon, AZ) attached to a frame grabber card under the control of Axon Imaging Workbench version 2.2 (Axon Instruments).
Electrodes were pulled using a horizontal pipette puller (model P-77, Sutter Instruments), lightly fire polished, and backfilled with filtered pipette solution [containing (in mM) 10 NaCl, 130 K-gluconate, 1.3 CaCl2, 4.1 MgCl2, 5 HEPES·KOH, and 5 EGTA (pH 7.4); 300 mosmol/kg]. Pipettes of optimal geometry had resistances of between 3 and 5 M when filled with this solution. The bath was grounded by the connection of an AgCl-coated silver wire via a 3% agarose "salt bridge" made with pipette solution. Current responses to voltage steps and ramps were usually filtered at 2 kHz using the inbuilt 4-pole Bessel filter and sampled at 10 kHz except in the case of capacitive transient analysis, where sampling was at 100 kHz with the antialiasing filter set at 10 kHz. Current records were analyzed offline using Clampfit version 9.0 (Axon Instruments). Curve fitting of capacitive transients were used to calculate membrane resistance (Rm), membrane capacitance (Cm), and Rs. Since membrane area is directly proportional to Cm and biological membranes have an approximate area capacitance of 1 µF/cm2 (26), cell membrane area (Am) was directly estimated from Cm (Am = Cm/k, where k = 1 µF/cm2). To enable comparison between fiber cells of vastly different membrane area, area-specific conductance (
s) was derived by relating total membrane conductance (gm = 1/Rm) to Am, thus giving values (in units of pS/µm2) of the fiber cell membrane (s = gm/Am, where Am is measured in units of µm2).
Quantification of s also allowed estimation of cell cable properties in a manner similar to that applied to neurons with elongated processes (26, 27). This geometric approximation describes how voltage partitions along the intracellular conductive route [intracellular resistivity (Ri; in /cm2 or /cm)] in the presence of parallel conductance pathways across the membrane (s) and depends directly on the cell (or process) diameter (d; in cm) and the ratio between membrane and intracellular resistivity per unit length. Thus,
 | (1) |
is the membrane electrotonic length constant. Ri has been estimated in rat lens fiber cells at 110 M/cm by impedance methods (1).
The relative permeability of membrane conductance to different anions was assessed by measuring their effect on current reversal potential (Erev), and the ratio of anion permeability to Cl– permeability (PX/PCl) calculated as follows:
 | (2) |
Whole lens dissection and electrophysiological recording.
Four posterior incisions were made in the extracted rat eyes to create four scleral flaps that were pinned out into a custom-made recording chamber that was transferred to the stage of a dissecting microscope (Stemi SV 11, Zeiss, Frankfurt, Germany). The pinned-out lens preparation was overlaid with warmed AAH (37°C) and continually perfused (2 ml/min) with AAH. The recording chamber (2 ml) was perfused using three solution reservoirs independently controlled by three-way taps via a manifold with 2 ml of downstream deadspace. Lenses were impaled with glass microelectrodes (150F-10, Clark Electromedical Instruments) manufactured using a pipette puller (P-77 Micropipette Puller, Sutter Instruments, Novato, CA) and had a resistance of between 3 and 5 M when backfilled with 1 M KCl. Microelectrodes were attached to the headstage of a microelectrode amplifier (TEV-200, Dagan, Minneapolis, MN). The bath was grounded by connection to an AgCl-coated silver wire 1 M KCl half-cell via a 3% agarose-1 M KCl salt bridge. Lens potential recordings were sampled at 1 Hz using a DigiData 1200B analog-to-digital converter (Axon Instruments) and recorded using Axoscope version 8.1 (Axon instruments) running on a Celeron 500-MHz PC under Windows 2000. The headstage was mounted on a micromanipulator (World Precision Instruments, Sarasota, FL) that was attached to the stage of the dissecting microscope. This electrophysiological setup was surrounded by a Faraday cage positioned on a vibration isolation table (Newport, Irvine, CA). Mean lens potential was determined at baseline and both before and after exposure to solutions of a varying ionic composition. The relative permeability of the lens to different anions (PX/PCl) was assessed by measuring their effect on membrane potential and calculated by substitution of 
Vm for Erev in Eq. 2. Summarized data were plotted in Microsoft Excel (Microsoft, Redmond, WA), and statistical analysis was performed using Origin version 7.5 (Originlab, Northampton, MA).
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RESULTS |
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s, and a more hyperpolarized Erev compared with cells incubated in either normal AAH or AAH in which Ca2+ had been removed (Table 1). Thus, the ability of Gd3+ to prevent vesiculation of isolated fiber cells is also associated with a generalized improvement in electrical properties of isolated fiber cells, suggesting that the inhibition of the nonselective leak conductance prevents the Ca2+ influx responsible for the activation of the cytoplasmic Ca2+-dependent proteases known to initiate vesiculation of isolated fiber cells (6, 52, 53).
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s (Fig. 1B), and Erev (Fig. 1C) against fiber cell length indicated that these membrane parameters appeared to be related to fiber cell length. However, within this data set, there was a large variability in membrane parameters. This is highlighted by comparing the current profiles of two pairs of selected cells of similar length. The two short fiber cells presented display dramatically different membrane properties (Fig. 1, D–F) despite being of very similar length. One cell (Fig. 1D, 68 µm) exhibited a low s (0.47 pS/µm2) and a hyperpolarized Erev (Fig. 1F, –88 mV). In contrast, another short fiber cell of similar length (Fig. 1E, 60 µm) exhibited a very high s (3.21 pS/µm2), a linearization of the current-voltage (I-V) relationship, and a profound depolarization of Erev (Fig. 1F, –1 mV), which are parameters characteristic of the activation of a nonselective cation channel. A similar pattern was found when comparing longer fiber cells. While one cell selected from this length range (Fig. 1G, 357 µm) had a low s (0.096 pS/µm2) and a hyperpolarized Erev (Fig. 1I, –53 mV), another cell of similar length (Fig. 1H, 383 µm) had a high s (1.36 pS/µm2), a linearized I-V relationship, and a depolarized Erev (Fig. 1I, +0.4 mV), again indicative of an increase in nonselective cation permeability.
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s > gs) fiber cells exhibited markedly lower Rm and a higher s relative to s < gs fiber cells of the same length. These changes were associated with a depolarization of Erev, indicating a loss of membrane selectivity concomitant with their activation. Because of their similar membrane properties recorded in vitro to those measured in lento, the s < gs group can be looked at as representing cells with properties as close as possible to those measured in healthy intact lenses. In contrast, the activated s > gs group displayed leaky membrane characteristics similar to those observed in vesiculating fiber cells. By plotting Rm versus length, it can be seen that s > gs and s < gs cells overlap significantly on the Rm axis (Fig. 2B), underscoring why membrane resistance alone is a poor marker of fiber cell membrane integrity. The use of gs as a selection criterion for activated cells cleanly split the fiber cell population independent of fiber cell length, allowing us to focus on cells with physiologically relevant membrane properties (Fig. 2, C and D). To gain an overview of how membrane permeability changes as fiber cells elongate, we plotted Erev recorded from s < gs cells against cell length (Fig. 2D). The plot clearly illustrates that, despite the inherent variability in Erev, the observed values for Erev tended to cluster near the expected equilibrium potential values for either K+ (EK), Cl– (ECl), or nonselective conductances (ENSC). From this analysis, it is apparent that while the majority of cells of all lengths clustered around or between ECl and ENSC, only a subset of short fiber cells clustered near EK. Thus, cell elongation appears to be associated with a change in membrane properties, which can explain the observed variability in our data set.
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s, the transmembrane voltage at membrane extremities of such cells exhibited only a 0.5 e-fold decrease in command potential, indicating that space-clamp errors were not the source of the observed length-related trends in membrane parameters. This analysis also showed that recordings from the activated fiber cells were more affected by space-clamp errors, since the average electrotonic decay length () was half as long in this group of cells (Table 2), another objective reason for excluding this group of cells from further analysis.
Differentiation-dependent changes in membrane permeability of isolated fiber cells.
At the equator lens, epithelial cells differentiate and elongate to form fiber cells. This process establishes a gradient of progressively longer and older cells that extends from the periphery to the center of the lens. Previously, we have shown that the length of isolated fiber cells is essentially equivalent to their length measured in the intact lens in vivo, and, therefore, the length of an isolated fiber is indicative of its differentiation state (54). To investigate whether the observed changes in the membrane properties of isolated fiber cells were differentiation dependent, cells were binned into three arbitrary length categories: short fiber cells (<150 µm), medium fiber cells (150 > medium fiber cell > 300 µm), and long fiber cells (>300 µm). Using these length groups, average I-V relationships for each of the three s < gs groups were obtained and plotted as both absolute currents (Fig. 3 A) and currents normalized to membrane capacitance (INorm; Fig. 3B). The absolute current carried at all potentials by short fiber cells was found to be lower than for the two longer cell groups (medium and and long fiber cells), which were indistinguishable in terms of both the shape and magnitude of their I-V responses (Fig. 3A). This similarity between the longer cell groups was preserved when currents were normalized to membrane capacitance (Fig. 3B), with both medium and long fiber cell groups overlying one another. However, normalization of currents to membrane area revealed that short fiber cells conducted approximately threefold more current per unit area in the outward direction than longer cells and also possessed an inwardly activating current component that altered the characteristic outwardly rectifying fiber cell I-V relationship at potentials below –80 mV (Fig. 3B). The activation of an additional, inwardly activating conductance at potentials hyperpolarized to –80 mV (
EK) implies the presence of a conductance component in short fiber cells that is absent in longer fiber cells. The differences in I-V plots between shorter fiber cells and longer fiber cells (medium and long fiber cell groups), as shown in Fig. 3B, plus the wide variability in Erev observed among cells of all lengths (Fig. 2D) implies that the underlying channel population giving rise to these currents may be more diverse than previously thought. Indeed, the gross morphology of whole cell currents was often observed to differ substantially even among cells of very similar lengths and baseline membrane properties. To further investigate this diversity, pharmacological and ion substitution experiments were performed.
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EK). At potentials hyperpolarized to EK (Vm < –80 mV), the conductance becomes strongly voltage activated. In the outward direction, the effect of Ba2+ is variable on this cell group, as evidenced by the larger error in this region. The Erev and I-V relationship of this Ba2+-sensitive current in the short fiber cell group strongly indicates the inhibition of a Kir channel of a type similar to that reported in the lens epithelium in many species (47). The TEA-sensitive current present in short fiber cells was strongly outwardly rectifying, of relatively large magnitude, reversed near EK, and activated at depolarized membrane potentials (Fig. 6B). The shape of the averaged difference current and the magnitude of the Erev were compounded by the presence of non-TEA-sensitive cells within the data set, but the presence of an outwardly rectifying, epithelial-like K+ conductance was distinguishable on the basis of the I-V shape and Erev (47).
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Thus, the pharmacological characterization of K+ channels in isolated fiber cells has revealed that newly differentiated short fiber cells contain K+-selective currents, which are reminiscent of those reported in the lens epithelium by numerous investigators (for a review, see Ref. 47). As seen in epithelial cells, outwardly rectifying K+ channels and Kir channels in short fiber cells appear to exist in parallel with each other, but neither are present to the same extent in all cells. In contrast, longer, more-differentiated fiber cells were dominated by other conductances that were not inhibited by these classic K+ channel inhibitors, indicating that a shift away from a dominant K+ channel occurs as cells elongate.
Characterization of anion permeability in isolated lens fiber cells. Since the solubility of anion channel inhibitors was compromised by the presence of Gd3+ in the bathing medium (54), anion replacement experiments rather than pharmacological experiments were used to characterize the contribution of anion channels to the membrane properties of isolated fiber cells of different lengths. To initially determine the contribution anion channels make to overall membrane current, extracellular Cl– was replaced with the impermeant anion gluconate. While gluconate replacement resulted in the reduction of an outwardly rectifying conductance recorded from the majority of fiber cells from all length groups, the magnitude of whole cell current alteration on gluconate replacement varied considerably between cells (Fig. 7). Fiber cells from the short fiber cell group appeared to display the most variability in their response to the removal of extracellular Cl– (Fig. 7, A and B). Those SFCs that initially exhibited a large outwardly activating conductance displayed a large reduction in outward current upon the removal of extracellular Cl– (Fig. 7A), whereas other cells of similar lengths but with a different current profile displayed a more modest reduction in current upon replacement of Cl– with gluconate (Fig. 7B). In contrast, all longer fiber cells [from the medium (Fig. 7C) and long (Fig. 7D) fiber cell groups] consistently responded to the replacement of Cl– with gluconate with a large inhibition of the outwardly rectifying current.
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Characterization of nonselective permeability in isolated lens fiber cells. Although the inhibition of a nonselective cation conductance with Gd3+ was a prerequisite for obtaining viable isolated fiber cells, it is evident that a residual leak conductance remains in the presence of Gd3+, since in many isolated fiber cells Erev was depolarized from EK and ECl and clustered near ENSC (Fig. 2D). This contribution of nonselective leak conductance to overall membrane current is shown in Fig. 9. Membrane currents recorded from a long fiber cell were dominated by an outwardly rectifying anion conductance (Fig. 9A), as shown by its characteristic response to gluconate replacement (Fig. 9B); however, Erev of this cell was substantially depolarized from ECl (Fig. 9D). The subsequent replacement of extracellular Na+ with the impermeant cation NMDG (Fig. 9C) resulted in a hyperpolarization of Erev toward ECl (Fig. 9D), suggesting that both Cl– and nonselective cation conductance contribute to the membrane properties of this cell. A different cell, which was dominated by an outwardly rectifying anion conductance (Fig. 9E), also exhibited a minor inward current component that was abolished by Ba2+ (Fig. 9F). This indicates that a residual K+ conductance still persists in this cell in parallel with the fiber cell anion conductance. Interestingly, the replacement of extracellular Na+ with NMDG (Fig. 9G) reduced overall current and hyperpolarized Erev toward EK (Fig. 9H). Thus, it appears that longer fiber cells can also possess a nonselective cation conductance that is insensitive to Gd3+ and may contribute to the membrane properties of isolated fiber cells.
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DISCUSSION |
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The dominance of K+ conductances we observed in short fiber cells is not only consistent with their differentiation from equatorial epithelial cells known to express a variety of K+ channels (9, 10, 45, 49) but also with the finding that these differentiating cells contain active Na+/K+-ATPase (23). At the equator, epithelial cells and differentiating fiber cells exhibit a pump current density per area that is 20 times larger than that measured at the anterior pole (23). This equatorial concentration of Na+ pump activity is responsible for active Na+ efflux and sets the lens resting potential (37), whereas the elongation of fiber cells increases the surface area available for the extrusion of Na+ (22). Since the Na+ pump couples Na+ efflux to K+ uptake, it makes sense that these newly differentiated fiber cells initially retain a K+ conductance to facilitate the K+ recycling necessary to maintain pump activity. However, as fiber cell differentiation proceeds, it is evident that the contribution of K+ channels to overall fiber cell conductance (Fig. 6) and Na+/K+-ATPase activity (12) both diminish as cells elongate. Whether the decrease in channel and pump activity occurs in parallel remains to be determined, but a dissociation is possible since we have recently shown that the rat lens expresses an alternative K+ efflux pathway that is mediated by an electroneutral KCl cotransporter (8). Furthermore, a reduction in K+ permeability has been postulated to help drive the increase in cellular volume that occurs during the rapid phase of fiber elongation seen in explant models of fiber cell differentiation (4, 5).
While the differentiation of epithelial cells into fiber cells has long been thought to be associated with the downregulation of K+ conductance (for a review, see Ref. 37), the mechanism by which this is achieved is less clear. Are the K+ channels inactivated/degraded in longer fiber cells or are they still present but become diluted/overwhelmed by other conductances that are activated during the course of fiber cell differentiation? Our results tend to suggest the latter, since some fiber cells from medium/long fiber cell length groups were shown to contain a K+ conductance that appears to be buried beneath an outwardly rectifying lyotropic anion conductance and a nonselective cation conductance (Fig. 9). This sporadic detection of apparent Ba2+- and TEA-sensitive current components suggests that K+ channels can persist in longer cells, but these are often difficult to detect against a background of more dominant conductances that emerge during fiber cell differentiation.
Our finding that an anion conductance dominates the membrane properties of longer isolated fiber cells is consistent with earlier experiments that reported modulation of resting lens voltage by extracellular Cl– removal (1, 3, 15), a series of morphological studies on whole lens organs cultured under isosmotic conditions that were exposed to a variety of anion channel inhibitors (8, 40, 51, 55), and patch-clamp studies that previously detected anion channel activity in isolated fiber cells and excised membrane patches (54, 56). In this study, we demonstrate, for the first time, that isolated fiber cells and whole lenses share a common lyotropic anion selectivity sequence (Table 3). Our results therefore indicate that the Cl– conductance we identified in isolated fiber cells is not an artifact of the dissociation process but that it also contributes to setting of the resting voltage in the intact lens. In other cell types, this lyotropic selectivity sequence (NO3– > I– > Cl– >> gluconate) has been associated with both volume-sensitive anion conductances and members of the Ca2+-activated Cl– channel family (30). While in this study we have made no attempt to distinguish between these two families, a proportion of the variability in the observed basal activity and kinetics of this Cl– conductance observed in differentiating fiber cells (Fig. 7) could be potentially attributable to cell-to-cell variations in cell volume and/or intracellular Ca2+ levels. The ongoing identification of signaling pathways in the lens that mobilize intracellular Ca2+ (14) suggests that these Cl– channels may potentially be modulated to control the overall contribution of Cl– conductance to the electrical properties of the lens, again indicating that lens fiber cells contribute more to the overall properties of the lens than originally anticipated.
A subpopulation of isolated fiber cells displayed Erev values more depolarized than ECl in the presence of Gd3+, suggesting that a Gd3+-insensitive Na+-permeable leak pathway remains active at rest even in cells that display a physiologically relevant conductance (s < gs). Thus, individual isolated fiber cells appear capable of existing in a stable state in vitro with a variable level of basal Na+ leak. From the results shown in Table 2, it is apparent that this baseline level of cation channel activity is grossly elevated in isolated cells that undergo vesiculation. Lens depolarization and elevation of intracellular Na+, both signs of the activation of a cation conductance, have been observed in animal models of diabetic cataract (32, 33) and in the aging human lens (16). In the diabetic lens, the activation of these nonselective cation channels is associated with dramatic changes in membrane potential (33) and ionic homeostasis (32) that manifest in tissue damage in the lens cortex (7). In contrast, the changes observed in human lenses occur more gradually in an age-dependent manner over many decades of life but ultimately result in age-related nuclear cataract (16). Obviously, further study into the regulation of cation channels is required to shed further light on the changes in membrane permeability know to accompany these different types of lens cataracts.
In summary, the trends in ionic selectivity with length directly measured here in viable isolated fiber cells confirms the original predictions of Mathias et al. (36) in whole lenses. While epithelial cells and short fiber cells are dominated by K+ conductance, longer fiber cells become increasingly dominated by an anion conductance with a low level of concomitant cationic leak. As such, this study provides the first direct confirmation at the single cell level of the spatial differences in ionic permeability believed to drive the lens internal circulation system. Further characterization of the membrane conductances described in this study should shed new light on how membrane permeability is regulated in the normal lens and altered during cataractogenesis.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and E (
). G: a longer fiber cell (357 µm) that showed an outwardly rectifying whole cell current and had membrane properties (
, medium fiber cells (n = 16); 
), and gluconate (