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Expression of the Inwardly Rectifying K⁺ Channel Kir2.1 in Native Bovine Corneal Endothelial Cells

¹From the Departments of Cell and Developmental Biology, ²Ophthalmology and Visual Sciences, and ³Physiology, University of Michigan, Ann Arbor, Michigan.

	Abstract

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PURPOSE. To determine the presence of Kir2.1 channels in native bovine corneal endothelial (BCE) cells and assess their contribution to the resting membrane potential.

METHODS. RT-PCR and Western blot analysis were used to detect the expression of Kir2.1 mRNA and protein in native BCE cells. Whole-cell patch-clamp recording was used to characterize Kir2.1 currents in freshly isolated, single BCE cells, as well as in BCE cell clusters. The contribution of Kir2.1 channels to the membrane potential (V_m) was assessed by whole-cell recording in the zero-current clamp mode in the absence and presence of Ba²⁺.

RESULTS. RT-PCR analysis confirmed that Kir2.1 was expressed in the native BCE cells. Western blot analysis with native BCE cell protein and a polyclonal anti-Kir2.1 antibody revealed a 60-kDa band that was blocked by the corresponding synthetic Kir2.1 peptide. Both single BCE cells and BCE cell clusters exhibited an inwardly rectifying K⁺ (Kir) current that was dependent on the extracellular K⁺ concentration. The Kir current was blocked by external Ba²⁺ or Cs⁺ in a voltage- and concentration-dependent manner. In 5 mM K⁺ Ringer’s, the V_m of cell clusters averaged -40.0 ± 4.1 mV (n = 14) and in 140 mM K⁺ Ringer’s it depolarized to -7.4 ± 1.8 mV. Application of Ba²⁺ in 5 mM K⁺ Ringer’s produced a concentration-dependent depolarization of V_m, with 10 mM Ba²⁺ depolarizing V_m from -53.4 ± 4.8 mV to -27.8 ± 6.3 mV (n = 6).

CONCLUSIONS. Native BCE cells express functional Kir2.1 channels that help determine the membrane potential and probably also play a role in transendothelial transport.

In this study, we combined molecular biological, biochemical, and electrophysiological approaches to show that Kir2.1 channels are in fact expressed in native BCE cells, where they play an important role in setting the membrane potential. Some of these data have been reported in abstract form (Yang, et al. IOVS 2001;42:ARVO Abstract 2704).

	Methods

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Fresh BCE Cell Preparation
Bovine eyes were obtained from a local abattoir and rapidly transported to the laboratory in ice-cold HEPES-buffered Ringer’s (HR) solution (135 mM NaCl, 5 mM KCl, 10 mM glucose, 1.8 mM CaCl₂, 1.0 mM MgCl₂, and 10 mM HEPES, [pH 7.4]). Adhering extraocular tissue was removed from the globes by dissection. The corneas were excised and placed endothelial side up in a plastic holder.⁸ The endothelial surface was rinsed and subsequently incubated at 37°C for 30 minutes in cell isolation solution (135 mM N-methyl-D-glucamine [NMDG]-Cl, 5 mM KCl, 10 mM HEPES, 3 mM EDTA-KOH, 10 mM glucose, 3 mM cysteine, 0.06 mg/mL papain, titrated to pH 7.4 with NMDG-free base). Papain was chosen after preliminary experiments indicated that corneal endothelial cells were more viable when dispersed with papain, as opposed to trypsin or dispase II. The endothelial surface was then gently washed with HR solution and the endothelial cells dislodged from Descemet’s membrane by gentle rubbing with a silicone rubber spatula. The dislodged cells suspended in HR solution were collected. The cells from 2 to 30 corneas were pooled and used for electrophysiological recording, total RNA preparation, or whole-cell protein preparation.

Total RNA Isolation
Total RNA was extracted from freshly isolated native BCE cells (Trizol reagent; Life Technologies, Inc., Rockville, MD), according to the manufacturer’s instructions. RNA pellets were suspended in diethylpyrocarbonate-treated water and quantified by ultraviolet (UV) spectrophotometry.

RT-PCR Analysis
Total RNA isolated from freshly isolated native BCE cells was reverse transcribed with random decamers with reverse transcriptase (RetroScript; Ambion, Austin, TX) according to procedures outlined in the manufacturer’s instructions. PCR was performed with a primer set specific for Kir2.1 in different species. The forward primer designed from base pairs 848 to 872 of the bovine Kir2.1 coding region (GenBank AY052548; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD)⁶ was 5'-GYA ARC AGG ACA TYG ACA AYG CAG A-3', and the reverse primer from base pairs 1253-1227 was 5'-GGC TCT AGA GGT ACR CTK GCC TGG TTG -3'. The housekeeping gene, glyceraldehyde--phosphate-dehydrogenase (GAPDH), served as a control. The forward primer for GAPDH was 5'-GTG AAG GTC GGA GTC AAC G-3' from base pairs 113-131 of the human GAPDH sequence (GenBank AF261085); the reverse primer was: 5'-GAG ATG ATG ACC CTT TTG GC-3' from the region 468-439. The oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The PCR products were generated with DNA polymerase (SuperTaq-Plus; Ambion) and cycled 30 times for GAPDH or 40 times for Kir2.1 (1 minute at 94°C, 1 minute at 50°C, and 1 minute at 72°C), followed by a 7-minute extension at 72°C. The PCR products were separated by 1.5% agarose gel electrophoresis. The sequence of the Kir2.1 RT-PCR product was confirmed by DNA sequencing analysis. Sequencing was performed by the DNA Sequencing Core Facility at the University of Michigan.

Transfection
The human embryonic kidney cell line HEK 293 expressing simian virus (SV) 40 large T antigen was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The expression construct pcDNA3.1/Kir2.1/green fluorescent protein (GFP) was generated by inserting the Kir2.1 coding sequence (GenBank AY052548)⁶ in-frame into the pcDNA 3.1/GFP, with the GFP tag fused to the 3' end of the Kir2.1. HEK 293 cells were transfected with 0.8 µg/mL of expression plasmid cDNAs (pcDNA3.1/GFP or pcDNA3.1/Kir2.1/GFP) using a lipophilic transfection agent in serum-free medium (LipofectAmine 2000 and Opti-MEM I, respectively; Life Technologies, Inc., Rockville, MD) according to the manufacturer’s protocol. Whole-cell extracts were prepared for Western blot analysis 72 hours after transfection. Freshly dissociated cells were used for electrophysiological recording 48 to 72 hours after transfection.

Western Blot Analysis
Whole-cell lysates from freshly isolated native BCE cells (40 µg/lane) or from HEK 293 cells expressing Kir2.1 (5 µg/lane) were subjected to a 4% to 20% linear gradient Tris-HCl gel (Ready Gel; Bio-Rad Laboratories, Hercules, CA). After electrophoresis, proteins were transferred to nitrocellulose membrane and immunoblotted with affinity-purified rabbit polyclonal anti-Kir2.1 antibody (Alomone Laboratories, Jerusalem, Israel) at a dilution of 1:400, followed by development with enhanced chemifluorescent (ECF) substrate (Amersham Pharmacia Biotech, Piscataway, NJ) and chemifluorescence visualization using a phosphorescence imager (Phosphorimager; Molecular Dynamics, Sunnyvale, CA).⁹ The specificity of anti-Kir2.1 antibody staining was assessed by peptide blocking studies in which the antibody was incubated with the Kir2.1 peptide antigen (Alomone Laboratories) before immunoblot analysis.

Electrophysiological Recordings
Whole-cell recordings were performed with standard techniques, essentially as described by Hughes and Takahira.¹⁰ Freshly isolated native BCE cell clusters (5–20 cells) or individual single cells were placed in a continuously perfused Lucite recording chamber. Cells selected for recording had a bright appearance under phase-contrast microscopy. Except where noted, all experiments were conducted at room temperature (23–25°C). Patch pipettes were pulled from glass tubing (7052; Garner Glass, Claremont, CA) with a multistage programmable puller (Sutter Instruments, San Rafael, CA) and heat polished to resistances in the range of 3 to 5 M just before use. Voltage-clamped currents were recorded by whole-cell patch-clamp recording with an amplifier (Axopatch 1D; Axon Instruments, Foster City, CA). The average membrane capacitance (C_m) of single cells was 7 ± 3 pF (±SD, n = 13). C_m of cell clusters was roughly proportional to the number of cells within the cluster. For four clusters containing 18 ± 3 cells (range: 14–20), C_m averaged 107 ± 13 pF. Both voltage-clamp and zero-current clamp data were acquired and analyzed on computer (pCLAMP 8 software; Axon Instruments, Union City, CA). Membrane potential (V_m) was measured in the whole-cell recording mode under zero-current clamp. All voltages were corrected for an offset potential resulting from the liquid junction potentials between the pipette tip and bath solution, which averaged -10 mV. Series resistance averaged 11.8 ± 4.5 M (±SD, n = 13) and was not compensated.

It is possible that cells within clusters were not voltage clamped to the same value because of space-clamp limitations. This effect would be greatest at voltages farthest removed from the reversal potential and could lead to an underestimation of the degree of inward rectification. However, this problem is not relevant to measurements of open-circuit potential or reversal potential.

Solutions
The standard bath solution was HR (described earlier). In experiments testing the effects of extracellular K⁺ concentration on current or V_m, a portion of NaCl in HR was replaced with KCl to achieve [K⁺] + [Na⁺] = 140 mM. In blocking experiments, BaCl₂ or CsCl₂ was added to the bath solution to the final concentrations indicated. Bicarbonate-buffered Ringer’s consisted of 120 mM NaCl, 5 mM KCl, 27.5 mM NaHCO₃, 10 mM glucose, 1.8 mM CaCl₂, 1.0 mM MgCl₂ (pH 7.4) and was bubbled with 5% CO₂-95% O₂. The osmolality of all external solutions was 288 ± 5 mmol/kg, except when they contained 10 mM BaCl₂ or CsCl, in which case, solutions had osmolalities of 320 ± 7 and 312 ± 4 mmol/kg, respectively. Control experiments using isotonic solutions in which BaCl₂ or CsCl were substituted for NaCl confirmed that changes in current and membrane voltage were due to the blockage of ion channels as opposed to cell shrinkage.

The pipette solution consisted of 30 mM KCl, 83 mM potassium gluconate, 5.5 mM EGTA-KOH, 0.5 mM CaCl₂, 4 mM MgCl₂, 10 mM HEPES, and 4 mM K₂ATP and was titrated to pH 7.2 with KOH. The osmolality of the pipette solution was 244 ± 5 mmol/kg.

	Results

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RT-PCR Analysis of Kir2.1 Expression
Figure 1 demonstrates the presence of Kir2.1 mRNA transcript in native BCE cells. RT-PCR of native BCE mRNA with a primer pair specific for Kir2.1 resulted in a single band of the expected size (406 bp), identical with the predominant band that resulted from cloned Kir2.1 cDNA. The identity of the product was confirmed to be Kir2.1 by DNA sequencing. No band was observed in a reaction of template and primers without reverse transcriptase (minus RT), indicating that the 406-bp band was generated from mRNA rather than contaminating genomic DNA. RT-PCR with PCR primers for GAPDH also resulted in a band of the expected size, establishing the integrity and viability of the RNA.

View larger version (60K): [in this window] [in a new window]   FIGURE 1. RT-PCR analysis of Kir2.1 expression in native BCE cells. Total RNA isolated from native BCE cells was treated with DNase I, then reverse transcribed with random decamers. PCR was performed using Kir2.1-specific primers (yielding a predicted PCR product of 406 bp) or GAPDH-specific primers (yielding a predicted PCR product of 356 bp). Cloned Kir2.1 cDNA: the positive control PCR reaction.

View larger version (63K): [in this window] [in a new window]   FIGURE 2. Western blot analysis of Kir2.1 expression in native BCE cells. (A) Whole-cell lysates (40 µg/lane) from native BCE (A, lanes 1, 2) were subjected to a 4% to 20% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-Kir2.1 antibody (lane 1) and anti-Kir2.1 antibody preabsorbed with synthetic peptide (lane 2). Kir2.1 protein (60 kDa; lane 1) was resolved with a polyclonal anti-Kir2.1 antibody at a dilution of 1:400. The 60-kDa band was blocked by the synthetic Kir2.1 peptide used to generate the antibody (lane 2). (B) Whole-cell lysates (5 µg/lane) from HEK 293 cells transfected with pcDNA3.1/GFP (lane 1) or pcDNA3.1/Kir2.1/GFP (lanes 2, 3) were subjected to 4% to 20% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-Kir2.1 antibody (lanes 1, 2) and anti-Kir2.1 antibody preabsorbed with its synthetic peptide (lane 3). The anti-Kir2.1 antibody recognized an 80 kDa protein (GFP-tagged Kir2.1) in HEK 293 cells transfected with pcDNA3.1/Kir2.1/GFP (lane 2), but not in HEK 293 cells transfected with pcDNA3.1/GFP (lane 1). The 80-kDa band was blocked by the synthetic Kir2.1 peptide (lane 3).

Expression of Inwardly Rectifying K⁺ Current in Native BCE Cells
To evaluate native BCE cells for expression of functional Kir2.1 channels, we recorded whole-cell currents in single, acutely dissociated cells. Figure 3A shows families of currents recorded in a representative cell bathed in 5 mM K⁺ and 140 mM K⁺ Ringer’s. Figure 3B shows the corresponding current–voltage (I-V) relationships averaged from six cells. In 5 mM K⁺ Ringer’s the I-V relationship was essentially linear, but exhibited a somewhat larger slope conductance at voltages negative to about -80 mV. In contrast to previous reports on rabbit corneal cells,⁴ we did not observe transient or sustained outwardly rectifying currents, even when the membrane potential was held at -60 mV between voltage steps (not shown). When extracellular K⁺ concentration ([K⁺]_o) was increased to 140 mM, inward current increased dramatically and shifted the cell’s zero-current potential in the positive direction. Membrane hyperpolarization from a holding potential of -10 mV rapidly activated an inward current that was sustained except at strong negative potentials, when slow inactivation occurred. In six cells, increasing [K⁺]_o from 5 to 140 mM K⁺ increased the inward slope conductance measured between -140 and -150 mV from an average of 0.26 ± 0.12 to 0.76 ± 0.20 nS and depolarized V_m from -22.4 ± 3.0 to -3.13 ± 2.3 mV. Both of these K⁺-induced changes are consistent with the presence of inwardly rectifying K⁺ channels, but the relatively depolarized membrane potential in 5 mM K⁺ suggested that the membrane had a significant leak conductance. In addition, in most cells, a noisy current appeared after several minutes of recording that made further characterization of the inwardly rectifying K⁺ current difficult.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Inwardly rectifying K+ currents in single isolated BCE cells. (A) Whole-cell current recorded from a representative native BCE cell showing a large increase in inward current produced by elevating the extracellular K+ concentration from 5 (top) to 140 (bottom) mM. The horizontal line to the left of the current records indicates the zero-current level. The voltage protocol used to evoke these currents is shown below the panels. (B) Summary of current-voltage relationships obtained in six single BCE cells bathed in 5 and 140 mM K+. Data represent the mean ± SEM.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Inwardly rectifying K+ currents in BCE cell clusters. (A) Whole-cell current records from a representative native BCE cell cluster showing a large increase in inward current produced by increasing extracellular K+ concentration from 5 (top) to 140 (bottom) mM. The horizontal line to the left of the current records indicates the zero-current level. The voltage protocol used to evoke these currents is shown below the panels. (B) Summary of current-voltage relationships obtained in five BCE cell clusters bathed in 5 and 140 mM K+. Note the large increase in inward current and positive shift in zero-current potential (V0). In 5 mM K+, V0 ranged from -49 to -31 mV and in 140 mM K+, it ranged from -9.3 to -6.7 mV. Currents in each cell were normalized to the current at -160 mV in 140 mM K+.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Inwardly rectifying K+ currents in native BCE cells are blocked by external Ba2+. (A) Whole-cell currents recorded from a representative native BCE cell cluster bathed in 140 mM K+ Ringer’s and in 140 mM K+ Ringer’s plus 10 µM Ba2+. External Ba2+ induced a slow time-dependent block of inward K+ currents. (B) Current-voltage relationships of native BCE cell clusters in the presence of the indicated concentrations of Ba2+. Data represent the mean ± SEM in four BCE cell clusters. Currents in each cell cluster were normalized to the current at -160 mV measured in the absence of Ba2+.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Inwardly rectifying K+ currents in native BCE cells are blocked by external Cs+. (A) Whole-cell currents recorded from a representative native BCE cell cluster bathed in 140 mM K+ Ringer’s and in 140 mM K+ Ringer’s plus 100 µM Cs+. External Cs+ induced a rapid block of inward K+ currents. (B) Current-voltage relationships of native BCE cell clusters in the presence of the indicated concentrations of Cs+. Data represent the mean ± SEM for four BCE cell clusters. Currents in each cell cluster were normalized to the current at -160 mV measured in the absence of Cs+.

(1)

where I_B/I₀ is the ratio of currents measured in the presence and absence of the blocker and [B] is the blocker concentration. The K_d for the Ba²⁺-induced block at -100 mV averaged 1.2 ± 0.3 µM (mean ± SEM, n = 4). This value is comparable to those obtained from native Kir2.1 channels in cultured BCE cells (2.7 ± 0.4 µM at -100 mV)⁶ and cloned Kir2.1 channels expressed in Xenopus oocytes (2.6 ± 0.5 µM⁶ or 0·8 µM¹² at -100 mV).

In addition to being blocked by Ba²⁺, Kir2.1 channels are rapidly blocked by external Cs⁺ in a voltage- and concentration-dependent manner.^{6 11 13 14 15} Figure 6A shows the effect of Cs⁺ on whole-cell currents recorded from a representative BCE cell cluster bathed in 140 mM K⁺ Ringer’s. In the presence of 100 µM Cs⁺, inward currents at large negative potentials exhibited a rapid block, consistent with the fast kinetics of Cs⁺ binding and unbinding in Kir2.1 channels.^{11 13 16 17} Steady state I-V relationships obtained in the absence and presence of Cs⁺ are shown in Figure 6B . As the membrane was hyperpolarized, the fraction of current blocked by Cs⁺ increased, suggesting that Cs⁺ blocks the channel by binding to a site within the channel pore. Figure 6 also shows that the extent of current inhibition was dependent on Cs⁺ concentration. The K_d for the Cs⁺-induced block at -100 mV averaged 206.6 ± 66.1 µM (n = 4). This value is similar to those obtained previously from native Kir2.1 channels in cultured BCE cells (96.8 ± 19.0 µM at -100 mV)⁶ and cloned Kir2.1 expressed in Xenopus oocytes (69.9 ± 8.9 µM at -100 mV).⁶

Taken together, the sensitivity of the inwardly rectifying K⁺ current to extracellular Ba²⁺ and Cs⁺ suggest that it is mediated by Kir2.1 channels.

Impact of Kir Channels on the Resting Membrane Potential
To evaluate the contribution of Kir channels to the membrane potential (V_m), we recorded V_m from native BCE cell clusters in whole-cell configuration under the zero-current clamp condition while testing the effects of elevated [K⁺]_o and K⁺ channel blockers. Figure 7A depicts the V_m response of a representative BCE cell cluster to increases in [K⁺]_o. Increasing [K⁺]_o from 5 to 140 mM caused a significant depolarization, indicating that V_m was determined in part by a K⁺ conductance. Figure 7B summarizes the results of similar experiments on 14 native BCE cell clusters. In the presence of 5 mM K⁺, V_m averaged -40.0 ± 4.1 (mean ± SEM; range: -23 to -73 mV) and in the presence of 140 mM K⁺, it averaged -7.4 ± 1.8 (range: -18 to +2.4 mV). To obtain a quantitative estimate of the contribution of Kir conductance to V_m, we applied the chord conductance equation

(2)

where g_K is the inwardly rectifying K⁺ conductance, g_T is the total conductance, 1 - g_K/g_T is residual conductance normalized to the total conductance, E_K is the K⁺ equilibrium potential, and E_r is the reversal potential of the residual current. Assuming that 10 mM Ba²⁺ blocks g_K specifically and completely, E_r can be taken to be -28 mV (discussed later). This value of E_r is close to the equilibrium potential for Cl^- (-32.5 mV), suggesting the residual current is primarily Cl^- current. We used this value and equation 2 to calculate that the relative Kir conductance (g_K/g_T) of BCE cells bathed in 5 mM K⁺ averages 0.22 and can be as high as 0.81. The depolarization of V_m to near 0 by 140 mM K⁺ can be accounted for by a K⁺-induced increase in g_K, combined with the depolarization of E_K to 0 mV.

View larger version (11K): [in this window] [in a new window]   FIGURE 7. K+ conductance contributes to the membrane potential of native BCE cells. (A) Membrane potential (Vm) recorded from a representative native BCE cell cluster exposed to two concentrations of K+ (5 and 140 mM). Elevated [K+]o caused significant depolarization, indicating that K+ conductance contributes to Vm. (B) Summary of Vm obtained in native BCE cell clusters bathed in 5 and 140 mM K+. In the presence of 5 mM K+, Vm ranged from -73 to -23 mV and in the presence of 140 mM K+, it ranged from -18 to +2.4 mV. Data represent the mean ± SEM in 14 native BCE cell clusters.

View larger version (12K): [in this window] [in a new window]   FIGURE 8. Ba2+ depolarizes the membrane potential of native BCE cells in a concentration-dependent manner. (A) Vm recorded from a representative native BCE cell cluster bathed in 5 mM K+ Ringer’s and in 5 mM K+ Ringer’s plus the indicated concentrations of Ba2+. Ba2+ produced a concentration-dependent depolarization of Vm. (B) Summary of Vm obtained in six native BCE cell clusters bathed in 5 mM K+ Ringer’s and in 5 mM K+ Ringer’s plus the indicated concentrations of Ba2+. Application of Ba2+ in the presence of 5 mM K+ produced a concentration-dependent depolarization of Vm, with 10 mM Ba2+ depolarizing Vm from -53.4 ± 4.8 mV to -27.8 ± 6.3 mV.

	Discussion

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In this study, we used RT-PCR and Western blot analyses to show that native BCE cells express Kir2.1, an inwardly rectifying K⁺ channel subunit that is widely expressed in excitable and nonexcitable cells.^{15 18} We also obtained functional evidence for Kir2.1 channels in electrophysiological recordings from single BCE cells and isolated BCE cell clusters, which exhibited an inwardly rectifying K⁺ conductance with biophysical properties and a blocker sensitivity profile consistent with the Kir2.1 channel. This channel is a major component of the BCE K⁺ conductance and appears to be an important determinant of the membrane potential. These results extend our previous observation that functional Kir2.1 channels are expressed in cell cultures of BCE cells.

Molecular Evidence for Kir2.1 Channel Subunit Expression in Native BCE Cells
In the vertebrate eye, Kir2.1 mRNA has been detected in chick, rabbit, and human lens epithelium,¹⁹ mouse retina,²⁰ native rabbit corneal epithelium and endothelium,⁷ and cultured BCE cells.⁶ In this study, we detected a 406 bp RT-PCR product in native BCE cells using Kir2.1-specific primers. This band is unlikely to be the result of genomic DNA contamination because no DNA was amplified in absence of reverse transcriptase (Fig. 1) . Hence, we conclude that the product was amplified from Kir2.1 mRNA.

To examine Kir2.1 expression at the protein level, we performed Western blot analysis with a polyclonal antibody raised against human and bovine Kir2.1. We detected a 60 kDa band in native BCE cells and confirmed its identity as Kir2.1 by peptide blocking experiments (Fig. 2) . This molecular weight is larger than that expected for unmodified Kir2.1 (48.3 kDa), probably because of posttranslational modification. Kir2.1 possesses several potential glycosylation sites as well as putative phosphorylation sites for protein kinase A (S425), protein kinase C (S3, T6, S357, and T383), and tyrosine kinase (Y242 and Y 366).^{6 11 19 21 22}

Electrophysiological Evidence for Kir2.1 Channel Expression in Native BCE Cells
Whole-cell recording from both isolated single cells and small clusters of native BCE cells revealed an inwardly rectifying current that increased markedly when extracellular [K⁺] was elevated (Figs. 3 4) . Associated with this current increase was a positive shift in zero-current potential, indicating that the change in current amplitude was at least in part the result of an increase in K⁺ conductance. This behavior is qualitatively similar to that of Kir2.1 channels and other members of the Kir channel family, with conductance activated by increases in extracellular K⁺ concentration.^{6 11}

In addition to being activated by extracellular K⁺, Kir channels are sensitive to voltage-dependent blockage by extracellular Ba²⁺ and Cs⁺. We found that the inwardly rectifying K⁺ current in native BCE cells was blocked by external Ba²⁺ in a voltage- and concentration-dependent manner with a K_d at -100 mV of 1.2 ± 0.3 µM. This value compares well with the K_d for the Ba²⁺-induced block of cloned Kir2.1 channels^{6 12 23} and native Kir2.1 channels in different cell types,^{24 25} including cultured BCE cells,⁶ but it also agrees with the K_d’s of two other members of the Kir2 channel subfamily, Kir2.2 and Kir2.3.^{26 27 28 29 30} Members of the Kir2 channel family, however, can be distinguished on the basis of their sensitivity to Cs⁺. Compared with Kir2.2^{26 27} and Kir2.3^{28 29 30} channels, Kir2.1 channels possess an affinity for Cs⁺ that is about two orders of magnitude lower.^{11 16 17} The Kir conductance observed in native BCE cells had K_d of 207 ± 66 µM at -100 mV, which is in accordance with the low sensitivity of Kir2.1 channels. Taken together, the pharmacology of the Kir conductance in native BCE cell clusters suggests that they are composed of Kir2.1 channels.

To our knowledge, the present study is the first to identify Kir2.1 currents in native corneal endothelial cells. Rae and Shepard⁷ demonstrated the presence of Kir2.1 mRNA in native corneal endothelial cells from a variety of species, but failed to observe Kir2.1 currents. They argued that Kir2.1 protein expression could be as low as several copies per cell and therefore would be difficult to detect. In this study, we were able to resolve inwardly rectifying K⁺ currents in single isolated BCE cells. We can arrive at a rough estimate of the number of channels in these cells by dividing the leak-corrected macroscopic conductance observed in 140 mM K⁺ (0.5 nS) by the single-channel conductance (20–25 pS).^{11 31} Assuming a channel-open probability of 1, this approach yields an estimate of 20 to 25 channels per cell. Because open probability almost certainly is lower, this number is likely an underestimate.

Previously, Watsky et al.⁴ and Rae et al.⁵ described two types of Ba²⁺-insensitive K⁺ channels expressed in rabbit corneal endothelial cells: a transiently activated, outward rectifying K⁺ channel⁴ and a temperature- and anion-stimulated K⁺ channel.⁵ The latter exhibits mild inwardly rectifying unitary currents, but outwardly rectifying macroscopic currents due to an increase in open probability with depolarization.³² In the present study on native BCE cells, we did not observe either current. The reason for these differences between rabbit and BCE cells is unclear, but it may reflect a species-specific difference in gene expression.

Physiological Roles of Kir Channels in Native BCE Cells
In other cell types, Kir channels play a fundamental role in intracellular homeostasis and in the generation of the resting membrane potential.^{11 15 16 25 33 34} Our patch-clamp recordings from freshly isolated clusters of native BCE cells indicated a resting V_m of -40 ± 4 mV, consistent with previous measurements in corneal endothelial cells using microelectrodes^{35 36} and patch pipettes.^{32 37 38} These values, however, are significantly depolarized from the K⁺ equilibrium potential, indicating that other channels, such as nonselective cation channels³² and Cl^- channels,^{38 39 40} also contribute to the membrane potential. Nonetheless, we estimate that Ba²⁺-sensitive K⁺ channels contribute approximately 20% of the whole-cell conductance when BCE cells are bathed in 5 mM K⁺ solution. The current–voltage relationships shown in Figures 3 and 4 indicate that Kir2.1 is a dominant K⁺ channel in BCE cells and support the idea that it underlies the Ba²⁺-sensitive K⁺ current. Thus, we conclude that the Ba²⁺-induced depolarization of membrane potential is due to the blockage of Kir2.1 channels. We cannot exclude the possibility, however, that BCE cells may also contain a different type of Ba²⁺-sensitive K⁺ channel that we were unable to detect.

The corneal endothelium is vital to the maintenance of corneal clarity. The corneal stroma tends to swell due to the imbibition pressure produced by an abundance of proteoglycans,⁴¹ and this process is counteracted by the endothelial fluid pump, which secretes fluid into the aqueous compartment. A substantial portion of this fluid transport is coupled to active Cl^- transport, which is mediated by entry across the basolateral membrane through the Na⁺/K⁺/2Cl^- cotransporter and exits across the apical membrane through Cl^- channels.^{3 40 41 42} In addition, active HCO₃^- secretion generated by influx through a basolateral membrane Na⁺/2HCO₃^- cotransporter and efflux across the apical membrane via the Cl^-/HCO₃^- exchanger and anion channels is also important. Quinidine, a potent blocker of both Kir2.1⁴³ and a transient outwardly rectifying K⁺ channel,⁴ has been shown to elicit a swelling rate in corneas similar to that produced by ouabain,⁴ suggesting that either or both of these K⁺ channels play a major role in the active transport processes underlying the fluid pump in corneal endothelium.

The secondary active transport of Cl^- and HCO₃^- is ultimately driven by the transmembrane electrochemical gradient for Na⁺ that is established by the basolateral membrane Na⁺,K⁺-adenosine triphosphatase (ATPase). As in other cells, Na⁺,K⁺-ATPase activity is critically dependent on the presence of an efflux pathway to recycle K⁺, and Kir2.1 channels are likely to function in this regard. Another way K⁺ channels may be linked to the fluid pump is by affecting the electrochemical driving forces on Cl^- and HCO₃^- transport. Because Kir2.1 channels contribute to the membrane potential, their hyperpolarizing influence promotes the efflux of Cl^- and HCO₃^- through anion channels in the apical membrane.^{3 40 41 42} Ba²⁺-sensitive K⁺ channels have also been implicated in cell volume regulatory responses of BCE cells to hypotonic challenge, where they provide a pathway for the obligatory efflux of cations that accompanies Cl^- efflux through swelling-activated anion channels.³⁶ Our results suggest that the K⁺ channels involved in this process may be Kir2.1.

Taken together, this work provides the first evidence that native BCE cells express functional Kir2.1 channels. These channels help determine the membrane potential and may also play a role in transendothelial ion transport and cell volume regulation.

	Footnotes

 
Supported by National Eye Institute Grants EY11793 (SAE) and EY08850 (BAH) and Core Grant EY07703.

Submitted for publication December 19, 2002; revised March 17, 2003; accepted March 28, 2003.

Disclosure: D. Yang, None; D.K. MacCallum, None; S.A. Ernst, None; B.A. Hughes, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Bret A. Hughes, Room 420, Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105-0714; bhughes{at}umich.edu.

	References

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