______29JE12A Involvement of potassium channel in AQP1-mediated water and gas transport in erythrocytes Siddhartha G. Jena* Harvard College, Cambridge, Massachusetts, USA Diet, lifestyle, and genetics contribute to a staggering 100 million people, nearly a third of the population, suffering from elevated blood cholesterol levels in the USA. Though cholesterol is essential for numerous physiological functions, it is well documented that the long-term effects of elevated levels of plasma cholesterol pose a significant health risk and are causal factors for diseases including angina, cardiovascular disorders and diabetes, resulting in more deaths than cancer and HIV combined. The short-term effect of elevated plasma cholesterol is, however, little known. Our earlier studies report that elevated plasma membrane
cholesterol is detrimental to water and CO2 transport in erythrocytes or red blood cells (RBC), and that this transport is mediated via the water channel AQP1. In the current study,
the dysfunction of active water and CO2 transport via the AQP1 channel through the RBC membrane is further confirmed. Since the K+ channel is known to interact with AQP1, involvement of the K+ channel and other components associated with AQP1 at the RBC membrane was further explored. Results from the study demonstrate that vH-ATPase, the Cl– channel and the K+ channel are associated with and regulate AQP1 function at the RBC membrane, and that the chloride channel precedes the potassium channel in the AQP1- regulating signalling cascade. Furthermore, the K+ channel inhibitor Glyburide, currently in use to treat Type 2 diabetes, is capable of reversing the detrimental effects of elevated plasma membrane cholesterol on RBC function, suggesting its potential use in therapy. Keywords: AQP1, erythrocyte, cholesterol, Glyburide, K+ channel
1. INTRODUCTION detrimental effects of cholesterol was further Elevated plasma cholesterol affects nearly 100 million demonstrated from that study (Jena and Lee, 2010). Americans, a third of the US population, resulting in In the past decade, the tetradecapeptide wasp venom devastating health consequences and nearly one million mastoparan, GTP, the GTP-binding heterotrimeric Gαi/ + + – deaths annually. Although the long-term effect of Gαo proteins, vH -ATPase, PLA2, the K and Cl elevated plasma cholesterol is known for its accretion in channels, and aquaporins (Abu-Hamdah et al., 2004), arterial walls, little was understood of its immediate have been implicated in the gating of water into secretory effects until recently (Jena and Lee, 2010). Cholesterol in vesicles. A similar mechanism operating at the RBC blood plasma is known to rapidly enter and incorporate membrane is therefore hypothesized. Gαi3 has been into the red blood cell (RBC) plasma membrane (Tall, implicated in various tissues to regulate both K+ and Cl– 1993; Glomset et al., 1995; Ishizake et al., 1994). As early ion channels at the plasma membrane (Delport et al., as the 1970s, it was demonstrated in human studies that 1997; Knepper and Inoue, 1994, 1997). PLA2 has also blood plasma and RBC membrane cholesterol levels are been found to regulate both K+ and Cl– ion channels. Since Gα and PLA2 are present at the RBC membrane inversely associated with the transmembrane O2 i3 diffusion rate of RBC (Steinbach et al., 1974): as (Mukherjee and Maxfield, 2004; Hillman et al., 2005), I cholesterol levels in the blood plasma and consequently in hypothesize the involvement of both Gαi3 and PLA2 in + the RBC membrane decrease, O diffusion in RBC the regulation of AQP1 activity. Furthermore, since K 2 – increases (Steinbach et al., 1974; Menchaca et al., 2004). and Cl ion channels are present at the RBC membrane Then in 2010 (Jena and Lee, 2010), we reported for the (Hillman et al., 2005), the involvement of Gαi3 in the + – first time that the immediate effect of elevated plasma regulation of K and Cl ion channels at the RBC membrane and its possible implication for AQP1-mediated water entry cholesterol is dysfunction of active water and CO2 transport via the AQP1 channel through the RBC were investigated. Results from my studies demonstrate + – membrane. The effect of the phospholipase A2 (PLA2) that K and Cl ion channels are associated with AQP1 at + inhibitor ONO-RS-082 (ONO) on ameliorating the the RBC membrane, and that the K channel inhibitor Glyburide currently used in treating diabetes reverses the * E-mail: [email protected] detrimental effects of cholesterol on RBC function.
Journal of Biological Physics and Chemistry 13 (2013) 12–17 © 2013 Collegium Basilea & AMSI Received 4 November 2012; accepted 30 March 2013 12 ______Ion channels regulate AQP1-mediated water and gas exchange S.G. Jena 13
2. MATERIALS AND METHODS Optimal doses of the water channel stimulator mastoparan (Mas), the water channel inhibitor HgCl , cholesterol RBC were isolated according to our published procedure 2 (CHOL), the chloride channel inhibitor diisothiocyana- (Jena and Lee, 2010). To determine the rôle of cholesterol tostilbene-2,2’-disulfonic acid (DIDS), the potassium on AQP1-induced rapid gating of water into RBC, channel inhibitor Glyburide (GLY) (Glucovance®; cholesterol was incorporated into the plasma membrane Bristol-Myers Squibb), and the vH-ATPase inhibitor of isolated RBC also according to our published Bafilomycin, were then used in the study. procedure (Jena and Lee, 2010). The transport of CO 2 BCECF-AM measurement of CO transport into RBC was determined using an established BCECF 2 across the RBC membrane: Freshly prepared and fluorescence assay (Fig. 1). The presence of AQP1 and washed RBC were labelled with the acetoxymethyl ester the regulatory components involved in its function at the of BCECF (BCECF-AM, Molecular Probes), by RBC membrane were determined using immunoblot incubation with 20 mM BCECF-AM in PBS at 22 °C for and immunoprecipitation assays (Fig. 2). Mastoparan is 20 min. External dye were removed by five washes (100 g, a G-protein stimulator, which has been demonstrated to 10 min), and the labelled RBC were maintained on ice stimulate AQP1-induced RBC swelling (Jena and Lee, until measurements were made. Examination of cells by 2010), and was used in the study. Real-time changes in fluorescence microscopy helped determine the uniformity RBC size (Figs 3–5) were determined using the of labelling, and the absence of leukocytes (which are established right-angle dynamic light scattering (DLS) larger and brightly labelled). The BCECF-labelled RBC technique (Jena and Lee, 2010). To discount the effect of suspension was either untreated or treated with cholesterol passive water transport, all DLS experiments to determine or HgCl and incubated for 15 min in a sealed flask the relative size dynamics of RBC were carried out in water. 2 containing CO2. BCECF fluorescence was monitored at Student’s t-test was used to quantitatively compare groups, an excitation wavelength of 485 nm and detected at 515 nm with significance deemed to be established at P < 0.01. using a Hitachi F-2000 spectrofluorimeter (Fig. 1). Isolation of rat red blood cells: RBC were Western blot analysis and immunoisolation of isolated according to a published procedure (Jena and AQP1-associated proteins at the RBC membrane: Lee, 2010). Briefly, male Sprague–Dawley rats weighing Isolated RBC were lysed using sonication and centrifuged 100–120 g were used for the study. Blood was collected for 1 h at 100 000 g to obtain the cell plasma membrane. by cardiac puncture immediately following CO2-induced The RBC plasma membrane, obtained as a pellet, was euthanasia of the animal. The blood sample was diluted in solubilized using a 1% w/v Triton–Lubrol mixture and 10 volumes of PBS, and spun at 120 g for 10 min at 4 °C. mixed with an equal volume of Laemmli sample preparation The resulting pellet was resuspended in 2 volumes of buffer, prior to the one-dimensional 10% SDS–PAGE, PBS and washed three times, followed by final electrotransfer to nitrocellulose and immunoblot analysis resuspension in 10 volumes of PBS prior to use. using specific antibodies to AQP1 (Santa Cruz Biotechnology Cholesterol reconstitution into the RBC Inc., Santa Cruz, California). To immunoisolate the RBC- membrane: RBC membrane was reconstituted with membrane AQP1 and associated proteins, the 1% w/v excess cholesterol using a published procedure (Jena and Triton–Lubrol-solubilized RBC membrane was immuno- Lee, 2010). Following incubation of RBC at room isolated using specific antibodies to AQP1 conjugated to temperature for 30 min in buffer containing 40 μM protein G–sepharose beads (Santa Cruz Biotechnology cholesterol (Sigma-Aldrich, St. Louis, Missouri), the cells Inc.). The resultant immunoisolates was resolved using were lysed using sonication and centrifuged for 1 h at 10% SDS–PAGE, electrotransfer to nitrocellulose and 100 000 g to obtain the cell plasma membrane. The RBC immunoblotting using a specific antibody against potassium plasma membrane, obtained as a pellet, was solubilized and chloride channels, and vH-ATPase. Ten micrograms using 1% w/v Triton–Lubrol mixture, and assayed for of total RBC membrane (RBCM) protein solubilized in 1% cholesterol and protein content (data not shown). Triton–Lubrol, and 100 μg of RBCM immunoprecipitated Cholesterol content was measured using an Amplex Red using AQP1-specific antibody (RBCM-IP-AQP1), were Cholesterol Assay Kit (Molecular Probes) according to resolved using 10% SDS–PAGE, followed by electrotransfer the manufacturers’ instructions. Protein concentrations to nitrocellulose membrane, and probed using vH-ATPase-, were assayed using the Pierce BCA protein assay kit KiR6.1 (potassium channel)-, and CLC3 (chloride channel)- (Thermo Scientific, Rockford, Illinois) (Smith et al., 1985). specific antibodies (Santa Cruz Biotechnology Inc.). Determination of the optimal dose of various Dynamic light scattering of RBC: Measurements compounds used for the regulation of RBC swelling of RBC size dynamics were determined using real-time, due to rapid water entry: Dose–response experiments right-angle light scattering in a Hitachi F-2000 spectrofluo- using various compounds used DLS to assess RBC volume. rimeter (Jena and Lee, 2010). Real-time scattered light
JBPC Vol. 13 (2013) ______14 S.G. Jena Ion channels regulate AQP1-mediated water and gas exchange intensities at excitation and emission wavelengths of 530 nm incubation of RBC in 40 μM CHOL (Jena and Lee, 2010). were used to measure the size dynamics of RBC. The These studies demonstrated the incorporation of amount of light emitted from the sample at the emission exogenous cholesterol into RBC membrane. Similarly, to wavelength is directly proportional to the surface area of determine the optimal dose of the various compounds to the RBC, which increases as the volume increases due to be used in the study, dose–response experiments were water being transported into the cell. In these performed. 40 μM Mas, which was previously demonstrated experiments, isolated RBC were suspended in water and (Jena and Lee, 2010) to maximally stimulate rapid water rapid changes in their size was monitored before and after entry into RBC, was used in the study. addition of HgCl2 (an AQP1 inhibitor) and CHOL, To determine the effect of high cholesterol on AQP1 followed by Mas exposure.1 Results are expressed in function in RBC, the transport of gases through the RBC arbitrary units and as percent light scattered over controls. membrane was determined (Fig. 1). In an earlier study it Student’s t-test was performed for comparison between had been demonstrated that the negative surface charge groups, with significance set at P < 0.05. of RBC, deduced from their zeta potential measured Order-specific blocker analysis to determine using a ZetaSizer instrument, became less negative the signalling pathway involved in AQP1 regulation following exposure to carbon dioxide (Jena and Lee, at the RBC membrane: The various combinations of 2010). When CO2 enters the cell, the carbonic acid the sequential application of 40 mM of either the chloride generated within results in the neutralization of the net or potassium channel inhibitors or the PLA2 inhibitor negative charge of the RBC membrane. The more CO2 (ONO-RS-082), on Mas-induced RBC water entry and enters, the more positive the membrane potential of the swelling were analysed. RBC were exposed to the RBC membrane becomes. Similarly, using the pH- chloride channel inhibitor DIDS followed by the sensitive dye BCESF (Fig. 1), the transport of CO2 through potassium channel inhibitor GLY, and to GLY followed by AQP1 at the RBC membrane was further demonstrated. DIDS. Similarly experiments were performed using The BCESF results demonstrate that the AQP1 inhibitor
DIDS preceding exposure of RBC to ONO-RS-082 and HgCl2 blocks CO2 entry, preventing RBC acidification. the reverse sequence; and exposure first to GLY followed by ON-ORS-082 and the reverse combination, to determine which of them comes before the other in the signalling cascade. Three separate experiments were performed for each combination, with 8–15 recorded points per experiment, which were averaged and represented with their standard deviations in the results (Fig. 5).
3. RESULTS AND DISCUSSION RBC from rat blood was isolated for the study and its purity determined by both light microscopy and scanning electron microscopy (data not shown). Both light and electron micrographs of the isolated RBC demonstrated an intact and homogeneous preparation. To discount the effect of passive water transport into or out of the RBC Fluorescence intensity [FI] (arbitrary units) due to osmosis, all dynamic light scattering (DLS) experiments to determine the rapid size dynamics of RBC Wavelength / nm were performed in water. Under these conditions and pH decrease = FI increase experimental time window, no change in nonstimulated or Figure 1. The pH-sensitive dye (BCESF: 2’,7’-bis-2-carboxyethyl- osmotic water transport was observed, as previously 5-6-carboxyfluorescein) used to demonstrate CO2 transport demonstrated (Jena and Lee, 2010). Furthermore, to through AQP1 at the RBC membrane. The AQP1 inhibitor HgCl blocks CO entry, preventing RBC acidification. determine whether exposure of RBC to CHOL results in 2 2 Similarly, cholesterol blocks the entry of CO2 into RBC. Data its incorporation into the RBC membrane, CHOL was represents one of ten experiments. measured in RBC plasma membrane following 30 min
1 Mastoparan (Mas) is an amphiphilic tetradecapeptide from wasp venom that stimulates Gαi3/Gαo proteins, by promoting the GTPase activity of the G protein (Cho et al., 2002).
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Similarly, CHOL blocks the entry of CO2 into RBC as effectively as does HgCl2. These results demonstrate that the increase in RBC membrane cholesterol is detrimental to CO2 entry into RBC, as demonstrated by the near abrogation of RBC membrane charge neutralization and acidification compared to control RBC. Next, the presence and effect of potassium (K+) and chloride (Cl–) ion channels, and vH-ATPase in the RBC membrane, and their association with the water channel AQP1, were determined (Fig. 2). Immunoblot and DLS (arbitrary units) immunoprecipitation assays demonstrated the presence and association of vH-ATPase, KiR6.1 (potassium channel), and CLC3 (chloride channel) with AQP1 at the RBC membrane (Fig. 2). Blockers of vH-ATPase, and Concentration / μM of the K+ and Cl– ion channels, on Mas-induced RBC Figure 3. Dose–response of the chloride channel inhibitor volume changes were further determined using DLS diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS), the (Figs 3–5). Studies using the chloride channel inhibitor potassium channel inhibitor Glyburide and the vHATPase (DIDS), the potassium channel inhibitor (GLY) and the inhibitor Bafilomysin on mastoparan-induced water entry into vHATPase inhibitor (Bafilomysin) on Mas-induced water RBC. Note the dose-dependent inhibition of water entry in presence of DIDS, as opposed to a dose-dependent stimula- entry into RBC demonstrated a dose–dependent tion of RBC water entry in the presence of Glyburide and inhibition of water entry in presence of DIDS, as opposed Bafilomycin. The result further confirms the presence of to a dose–dependent stimulation of RBC water entry in vH-ATPase and potassium and chloride channels at the RBC the presence of GLY and Bafilomycin (Fig. 3). These membrane, and their interaction in the regulation of AQP1. functional studies further confirmed the presence of vH- ATPase and potassium and chloride channels at the RBC * P < 0.01 membrane, and their interaction in the regulation of AQP1, as demonstrated through the immunoblot anlysis (Fig. 2). These studies (Fig. 4) further demonstrate that rapid water gating and consequent swelling of isolated RBC is impaired by high cholesterol and ameliorated by the potassium channel inhibitor GLY. Percent-increase in DLS intensity
MW / kDa
Figure 4. Rapid water gating and consequent swelling of isolated RBC is impaired by high cholesterol and ameliorated by the potassium channel inhibitor Glyburide. On the left is a representative experiment demonstrating a time-dependent increase in DLS of RBC suspensions as a result of swelling due to rapid water entry following exposure to mastoparan (Mas). Note inhibition of rapid water entry following exposure to the chloride channel blocker diisothiocyanatostilbene-2,2’- disulfonic acid (DIDS), and stimulation in the presence of the potassium channel blocker Glyburide (GLY). Note that the stimulatory effect of Glyburide persists even in the presence of excess membrane cholesterol (depicted in red, colour online). Data represents %-increase in dynamic light scattering (DLS) Figure 2. Potassium and chloride channels are associated with intensity from isolated RBC in the presence of various AQP1 at the RBC membrane. Ten μg of total RBC membrane modulators. Data is obtained from 3 experiments with 8–15 (RBCM) protein solubilized in 1% Triton–Lubrol, and 100 μg of points per experiment (*, P < 0.01). RBCM immunoprecipitated using an AQP1-specific antibody (RBCM-IP-AQP1), was resolved using 10% SDS-PAGE, To determine the signalling pathway of AQP1 followed by electrotransfer to a nitrocellulose membrane, and regulation at the RBC membrane, various application probed using vH-ATPase, KiR6.1 (potassium channel), and CLC3 (chloride channel)-specific antibodies. Note the sequences of 40 μM of either the chloride or potassium association of vH-ATPase, potassium and chloride channels channel inhibitors, or the PLA2 inhibitor, on Mas-induced with AQP1 at the RBC membrane. RBC water entry and swelling were carried out (Fig. 5).
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* P < 0.05 possible use of Glyburide and ONO-RS-082 as ** P < 0.001 therapeutic agents to ameliorate the detriment of elevated RBC membrane cholesterol on water and gas transport into and out of the cell.
scattering intensity 4. SUMMARY AND CONCLUSION Percentage increase in Percentage increase This study demonstrates that elevated cholesterol in the RBC plasma membrane impairs both water and CO Figure 5. The various combinations of the sequential effect of 2 40 μM of either chloride, potassium or PLA2 inhibitors on transport into the cell. With respect to the impact of mastoparan (Mas)-induced RBC water entry and swelling. cholesterol on molecular transport in RBC, these results Data represents %- increase over the control of RBC further confirm the initial findings of Steinbach et al. stimulated with Mas. Note that when RBC are exposed to DIDS (1974) involving oxygen transport, and provide not only followed by GLY as opposed to GLY followed by DIDS, only the former is significantly inhibitory, suggesting that in the further evidence for an AQP1-specific dysfunction signalling cascade involved in the G-protein-mediated AQP1 caused by cholesterol in RBC but also an understanding stimulation, the chloride channel precedes the potassium of the signalling cascade leading to function and channel. Similarly, if DIDS precedes exposure of RBCs to the dysfunction of AQP1. Through the further development PLA2 inhibitor ONO-RS-082 (ONO) it is stimulatory, as opposed to the reverse sequence. This suggests that PLA2 is of our understanding of this signalling cascade, we were downstream of the chloride channel. Finally, since both the also able to determine that the potassium channel blocker potassium channel inhibitor GLY and the PLA2 inhibitor ONO Glyburide and the PLA2 inhibitor ONO-RS-O82 are both are stimulatory; and RBC exposure first to GLY followed by able to ameliorate the detrimental effect of elevated ONO is more stimulatory than the reverse combination, it can be inferred that the potassium channel is upstream of PLA2. cholesterol on RBC function. Therefore, potassium and These results therefore suggest that in the signalling cascade PLA2 inhibitors could be used as potential drugs to involved in the G-protein-mediated AQP1 stimulation, the treat elevated cholesterol-induced impairment of RBC chloride channel precedes the potassium channel, followed by function. Glyburide is currently being used as a drug to PLA2. Data represent one of 3 separate experiments, with 8–15 value points per experiment that were averaged and treat type 2 diabetes, and therefore potentially could be represented with standard deviation (*, P < 0.05; **, P < 0.001). used to treat detriments resulting from elevated blood cholesterol. Similarly, the PLA2 inhibitor “Darapladib” is When RBC are exposed to DIDS followed by GLY, as currently under development by GlaxoSmithKline as an opposed to GLY followed by DIDS, only the former anti-atherosclerosis agent to treat coronary heart sequence was found to be significantly inhibitory, suggesting disease. Furthermore, since the vH-ATPase inhibitor that in the signalling cascade involved in G-protein Bafilomycin potentiates active transport of water into mediated AQP1 stimulation, the chloride channel precedes RBC, further studies are being carried out to determine the potassium channel. Similarly, if DIDS preceded its potential use as a drug to ameliorate RBC dysfunction exposure of RBCs to the PLA2 inhibitor ONO it was due to elevated plasma cholesterol. stimulatory, as opposed to the reverse sequence. This suggests that PLA2 is downstream of the chloride ACKNOWLEDGMENT channel. Finally, since both the potassium channel inhibitor GLY and the PLA2 inhibitor ONO are The author thanks Dr Jin Sook Lee of Wayne State stimulatory, and RBC exposure first to GLY followed by University for her guidance and for providing isolated rat ONO are stimulatory but the reverse combination is not, it RBC for the study. SGJ generated the idea and protocol may be inferred that the potassium channel is upstream for this project, and performed all the experiments of PLA2. These studies, therefore, demonstrate that in the presented in the article independently. There are no signalling cascade involved in the G-protein mediated conflicts of interest. This work was in part presented at regulation of AQP1 function the chloride channel the 2010 Detroit Science Fair, receiving Grand Prize in the precedes the potassium channel, followed by PLA2. The Medicine Category and at the San Jose Intel ISEF 2010, current findings build upon earlier studies (Abu-Hamdah receiving First Prize in the Medicine Category. The et al., 2004), in which the association of regulatory author was a 2012 Intel Finalist. Provisional Patent # US proteins such as G-proteins and PLA2 have been found to 61/460,286 has been granted as a result of this work. associate with AQP1. However, the key finding in the current study is the order-specificity of the signalling REFERENCES cascade in water and gas transport in RBC via the Abu-Hamdah, R., Cho, W-J., Cho, S-J., Jeremic, A., Kelly, M., AQP1 channel. This new knowledge supports the Ilie, A.E. and Jena, B.P. Regulation of the water channel
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