May 2002
Volume 43, Issue 5
Free
Retinal Cell Biology  |   May 2002
Effects of Protein Kinase C on Delayed Rectifier K+ Channel Regulation by Tyrosine Kinase in Rat Retinal Pigment Epithelial Cells
Author Affiliations
  • Olaf Strauss
    From the Institute for Clinical Physiology, Benjamin Franklin University Clinic, Free University, Berlin, Germany.
  • Rita Rosenthal
    From the Institute for Clinical Physiology, Benjamin Franklin University Clinic, Free University, Berlin, Germany.
  • Dennis Dey
    From the Institute for Clinical Physiology, Benjamin Franklin University Clinic, Free University, Berlin, Germany.
  • Julia Beninde
    From the Institute for Clinical Physiology, Benjamin Franklin University Clinic, Free University, Berlin, Germany.
  • Guido Wollmann
    From the Institute for Clinical Physiology, Benjamin Franklin University Clinic, Free University, Berlin, Germany.
  • Hagen Thieme
    From the Institute for Clinical Physiology, Benjamin Franklin University Clinic, Free University, Berlin, Germany.
  • Michael Wiederholt
    From the Institute for Clinical Physiology, Benjamin Franklin University Clinic, Free University, Berlin, Germany.
Investigative Ophthalmology & Visual Science May 2002, Vol.43, 1645-1654. doi:
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      Olaf Strauss, Rita Rosenthal, Dennis Dey, Julia Beninde, Guido Wollmann, Hagen Thieme, Michael Wiederholt; Effects of Protein Kinase C on Delayed Rectifier K+ Channel Regulation by Tyrosine Kinase in Rat Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2002;43(5):1645-1654.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Investigation of the regulation of delayed rectifier potassium channels by protein kinases in retinal pigment epithelial (RPE) cells.

methods. Membrane currents of cultured RPE cells were measured in either the perforated-patch or whole-cell configuration of the patch-clamp technique.

results. Different potassium channel blockers from animal venoms (charybdotoxin, hanatoxin, agitoxin, tityustoxin) were used to identify the delayed rectifying outward current in RPE cells as current through potassium channels, mainly composed of Kv1.3 subunits. Extracellular application of a tyrosine kinase blocker, such as genistein (100 μM), lavendustin A (10 μM), or herbimycin A (4 μM), almost completely abolished the delayed rectifier currents. Intracellular application of the tyrosine kinase pp60c-src (30 U/mL) through a patch pipette led to an increase of the delayed rectifier current consistent with an activation of the delayed rectifier channels by src subtype tyrosine kinase. Inhibition (with 1 μM chelerythrine) or activation (with 1 μM phorbol ester PMA) of protein kinase C (PKC) did not change the activity of delayed rectifier channels. Inhibition of PKC also did not change the effect of tyrosine kinase inhibition of delayed rectifier currents. However, in cells with stimulated PKC, inhibition of tyrosine kinase by genistein led to an increase of delayed rectifier currents. Intracellular application of pp60c-src (30 U/mL) in cells with stimulated PKC led to inhibition of delayed rectifier currents.

conclusions. Tyrosine kinase, of the src family, can activate or inhibit delayed rectifier channels composed of Kv1.3 subunits in RPE cells. The activity of PKC determines whether tyrosine kinase activates or inhibits these K+ channels.

The retinal pigment epithelium (RPE) interacts closely with the neuronal retina and helps to maintain retinal function. 1 2 Patch-clamp studies have documented several types of voltage-dependent potassium channels in the RPE: the inward rectifier, 3 4 5 6 7 8 9 10 M-type currents, 11 Ca2+-dependent potassium channels, 12 and delayed rectifier channels. 4 6 7 8 9 13 14 Immunocytochemical and PCR-based studies have demonstrated the expression of the outwardly rectifying potassium channel subunits Kv1.2, Kv1.3, Kv1.4, Kv2.1, and Kv4.2. 15 The functional role of the delayed rectifier channels remains unclear. 
The RPE secretes a variety of growth factors that help to maintain the structural integrity of photoreceptors, 16 17 18 19 and the RPE expresses a subtype of Ca2+ channel that is known to regulate secretion. 20 K+ channels often appear as functional antagonists of Ca2+ channels in regulating intracellular Ca2+ homeostasis. 21 Because L-type Ca2+ channels in RPE cells are regulated by tyrosine kinase of the src family, 22 the regulation of delayed rectifier channels by src kinase is of interest. 
Many Kv potassium channel subtypes are known to be regulated by tyrosine kinase. Direct interaction of tyrosine kinase and the K+ channel protein is likely in the regulation of the potassium channel subtypes Kv1.2, Kv1.3, Kv1.5, and Kv2.1. 23 24 25 26 27 The tyrosine kinase-dependent regulation of K+ channels is involved in cellular responses to muscarinic 24 and glutamatergic stimulation, 28 to stimulation of apoptosis 29 and proliferation, 30 31 32 and to stimulation by insulin 26 or growth factors. 33 34 However, tyrosine kinase sometimes has contrary effects on K+ channel activity. Ca2+-dependent K+ channels or Kv1.5 potassium channel subunits are activated or inhibited by activation of tyrosine kinase. 25 27 30 32 35 36 37 38 These discrepancies may be due to the different cell systems used, the effects of different kinases, or the interaction with other cellular ion channel-regulating mechanisms. 
The purpose of the study was to characterize the protein kinase-dependent regulation of the delayed rectifier potassium channel in RPE cells to obtain information about the role of this channel in RPE cell function. 
Materials and Methods
Cell Culture
Animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Primary cultures of rat retinal pigment epithelial cells were established according to the method of Edwards. 39 After enucleation, eyes were stored overnight in Puck’s saline F. 40 The eyes were incubated in Puck’s saline F without Ca2+ and Mg2+ and containing 0.1% trypsin for 35 minutes at 37°C. The bulbi were opened with a circumferential incision along the ora serrata to remove the anterior parts together with vitreous and retina. The RPE was gently brushed away from the Bruch membrane with a fine pair of forceps and collected in Ham’s F10 culture medium (supplemented with 20% fetal calf serum, 100 μg/mL kanamycin, and 50 μg/mL gentamicin). After trituration of the sheets of RPE cells by gentle pipetting, the cell suspension was plated into Petri dishes with glass coverslips. The cultures were maintained at 37°C and 5% CO2 in air, and the medium was changed twice a week. After 24 hours, the cells had settled down and started to spread out. Three- to 8-day old cultures were used for electrophysiological recordings. 
Patch-Clamp Recordings
Patch-clamp experiments were performed at room temperature. All experiments were performed in the perforated-patch configuration, except for experiments in which tyrosine kinase or genistein was applied intracellularly. Coverslips with RPE cells were placed in a perfusion chamber mounted on the stage of an inverted microscope. The cells were superfused with a bath solution containing (in millimolar): 0.3 CaCl2, 3 KCl, 0.6 MgCl2, 130 NaCl, 14 NaHCO3, 1 Na2HPO4, 33 HEPES, and 5.5 glucose, adjusted to pH 7.2 with Tris. Substances dissolved in dimethyl sulfoxide (DMSO) containing stock solutions were added to bath solutions so that the final DMSO concentration did not exceed 0.1%. This DMSO concentration has been shown not to influence membrane conductance. 41 Therefore, in experiments with concomitantly application of two DMSO-soluble substances, the concentration of one of these substances (in most cases, genistein) was reduced so that the DMSO concentration did not exceed 0.1%. Patch pipettes with a resistance of 3 to 5 MΩ were pulled from borosilicate tubes using a puller (DMZ Universal; Zeitz, Augsburg, Germany). The pipette solution contained (in millimolar): 0.5 CaCl2, 5.5 EGTA-KOH, 100 KCl, 2 MgSO4, 10 NaCl, and 10 HEPES, adjusted to pH 7.2 with Tris. For perforated-patch recordings, the pipette solution additionally contained 150 μg/mL nystatin. To avoid hyperosmotic swelling of the cells, the pipette solution was hyposmotic to the bath solution (approximately 60 mOsm) measured using a vapor pressure osmometer (model 5100B; Wescor, Logan, UT). With these solutions, no changes in cell size were observed during the whole-cell configuration. Whole-cell currents were measured with a patch-clamp amplifier ( EPC-9; Heka, Lamprecht, Germany) and low-pass–filtered at 3 kHz. Electrical stimulation and data storage and analysis were performed using a computer (TIDA hard- and software; Heka) in conjunction with an AT-compatible computer. 
The membrane capacitance and access resistance were compensated after the whole-cell configuration was established. The access resistance was compensated throughout the experiments to ensure that the remaining resistance did not exceed 10 MΩ. The resting potential of RPE cells was −40 ± 1 mV (n = 62); the membrane capacitance, 65.6 ± 6.6 pF (n = 62), and the access resistance, 17.0 ± 1.5 MΩ (n = 62; data for perforated-patch and whole-cell recording). The resting potential was measured directly after establishing the perforated-patch or the whole-cell configuration on cells that had not been compensated for membrane capacitance and access resistance, both of which were estimated after each experiment. The membrane capacitance was calculated from the integration of the area below a transient capacitative current curve, resulting from a voltage step of +10 mV for 30 ms. The access resistance was calculated from the relaxation time constant of the same capacitative current, where the ratio of the time constant to the membrane capacitance yields the access resistance. In perforated-patch recordings, the access resistance was approximately 10 MΩ higher than in whole-cell recordings. Maximum current amplitudes were measured at the end of a voltage step from −45 mV to +45 mV for 50 ms. 
If not stated otherwise, voltage-dependent currents were activated using a voltage-step protocol consisting of nine voltage steps of 10 mV increasing amplitude and 50-ms duration to depolarize cells from a holding potential of 45 mV. 
Intracellular Application of pp60c-src
The experiments on intracellular application of pp60c-src were based on those performed by Wang and Salter 42 and performed in the whole-cell configuration. For this purpose, the pipette solution additionally contained 4 mM Mg-adenosine triphosphate (ATP) and 30 U/mL pp60c-src. Because chloride currents are activated during the whole-cell configuration with physiological concentrations of ATP in the patch pipette, the bath solution contained an additional 1 mM of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The experiments were performed as follows: Establishment of the whole-cell configuration was followed by compensation of the membrane capacitance and access resistance, which usually took 60 seconds. The maximum current amplitude was then measured every 1 minute at the end of a voltage step from the holding potential of −45 mV to +45 mV of 50 ms duration. The current amplitudes were normalized to the maximum current amplitude measured 60 seconds after establishing the whole-cell configuration and plotted against time. Two sets of experiments were performed. For control experiments, the pipette solution contained either MgATP alone or MgATP and heat-inactivated pp60c-src (for heat inactivation, pp60c-src was boiled for 30 minutes at 95°C). The main experiments were performed with MgATP and active pp60c-src in the pipette solution. 
Preparation of Membrane Proteins
For isolation of membrane proteins, confluent RPE cell cultures in two culture flasks with 75 cm2 surface area were used. Cell lysis was performed in lysis buffer containing (in millimolar): 150 NaCl, 50 Tris, 1 Na-orthovanadate, and 0.05 NaF (pH 7.2, with HCl). The lysis buffer was supplemented with 1% Triton X-100, 0.5% Na-desoxycholate, 0.1 SDS, and 0.3 μg/mL EDTA. For protease inhibition lysis buffer contained (in micrograms per milliliter): 16 benzamidine-HCl, 10 phenanthrolene, 10 leupeptin, 10 pepstatin, 174 phenylmethylsulfonyl fluoride, and 1 aprotinin. Cell lysis was performed with a homogenizer (Polytron; Kinematik, Littau, Switzerland) and by three freezing and thawing steps (liquid N2; 42°C). Cell lysates were centrifuged at 5000 rpm for 5 minutes at 4°C. The supernatant was centrifuged a second time at 18,000 rpm for 30 minutes. The pellet was suspended in lysis buffer and subjected to Western blot analysis. 
Western Blot Analysis
Membrane proteins were separated by polyacrylamide gel electrophoresis (8.5% polyacrylamide). Equal amounts of protein were loaded in each lane of the gels (approximately 30 μg of total protein). Electrophoresis was performed in mini protean cells (Bio-Rad Life Science Group, Hercules, CA) for 1 hour at 150 V. The proteins were then blotted to nitrocellulose filter screens (Polyscreen; NEN Life Science Products, Boston, MA) for 1 hour at 100 V. Protein blots were blocked in PBS-Tween supplemented with BSA (5%) for 2 hours at room temperature. The same solution was used to dilute the antibodies (according to manufacturer’s instructions). Treatment with blocking peptides was performed according to the manufacturer’s instructions. The blots were probed overnight at 4°C with antibodies against Kv1.3 K+ channel α-subunits or phosphotyrosine. After washing (three times for 10 minutes in PBS-Tween), the blots were incubated with a 1:20,000 dilution of peroxidase-conjugated secondary antibody (Dianova; Jackson ImmunoResearch Laboratories, Hamburg, Germany) for 1 hour at room temperature. After final washing steps (three times for 10 minutes in PBS-Tween) the blots were visualized using a chemiluminescence kit (Amersham Pharmacia Biotech, Braunschweig, Germany) according to the manufacturer’s instructions. Blots were digitalized by an image analyzer (LAS-1000; Fujifilm, Berlin, Germany; with AIDA 2.0 software; Raytest, Berlin, Germany) in conjunction with an AT-compatible computer. 
Chemicals
Media and cell culture supplements were purchased from Gibco Life Technologies (Eggenstein, Germany). All chemicals were purchased from Sigma (Munich, Germany), Research Biochemicals (Cologne, Germany), Serva (Heidelberg, Germany), and Merck (Darmstadt, Germany). The potassium channel blockers and anti-Kv1.3 antibodies were purchased from Alomone Laboratories (Munich, Germany). Anti-phosphotyrosine antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). 
Statistical Analysis
Data are expressed as the mean ± SEM and were analyzed for significance by the unpaired Student’s t-test. Data were considered to be significantly different at P < 0.05. All electrophysiological recordings and Western blot experiments were performed 3 to 10 times. Unless otherwise stated, the figures show one representative experiment. 
The activation of delayed rectifier current was analyzed with the Hodgkin-Huxley n 2 model 43 :  
\[I{=}I_{\mathrm{max}}(1{-}\mathrm{e}^{{-}t/{\tau}_{\mathrm{act}}})^{2}\]
where I is the current at a given voltage and a given time, I max is the maximum current at this voltage, t is time, and τact is the activation time constant. 
Results
Outwardly Rectifying Current
Depolarization of RPE cells from a holding potential of –45 mV led to activation of voltage-dependent outwardly rectifying currents with delayed rectifier characteristics (Fig. 1) . The depolarization-induced currents activated at potentials more positive than –30 mV and reached at +45 mV a maximum amplitude with a current density of 3.6 ± 0.3 pA/pF (n = 4). The activation time constant was 8.0 ± 0.8 ms (n = 5; estimated by analysis with the Hodgkin-Huxley type n 2 model). We used several specific blockers to identify the Kv subtypes of K+ channels responsible for the outwardly rectifying currents (Fig. 2) . Application of charybdotoxin (10−7 M) led to a reduction of the currents to 68.2% ± 4.2% (n = 4). Agitoxin-2 (10−8 M), the blocker of Kv1.3 potassium channels, almost completely blocked the outwardly rectifying current (to 1.25% ± 1.25%; n = 4). The currents were insensitive to hanatoxin (10−7 M; blocker of Kv1.2 and 2.1 channels; 98% of control, recovery 92% of control; n = 1; data not shown) and tityustoxin Kα (10−8 M; blocker of Kv1.2 and 1.4 channels; 93.3% ± 3.9% of control). Thus, the outwardly rectifying currents in RPE cells were mostly K+ currents through Kv1.3 channels. 
Effect of Tyrosine Kinase on the Delayed Rectifier
The next set of experiments investigated the effects of chemically different tyrosine kinase blockers 44 (Fig. 3) . The experiments were performed in the perforated-patch configuration, because this configuration does not result in cell dialysis by the pipette solution with subsequent washout of regulatory enzymes, such as protein kinases. Extracellular application of genistein (100 μM) almost completely abolished delayed rectifier currents after 6 to 7 minutes. In the presence of genistein, the maximum amplitudes of the delayed rectifier current were 16.6% ± 13.3% of the control (50.5% ± 8.4% recovery; n = 5). Application of the inactive genistein analogue daidzein (100 μM, Fig. 4 ) did not lead to changes of the maximum current amplitude (98.8% ± 3.6% of control in the presence of daidzein; n = 3). The same was observed using lavendustin A (10 μM). Application of lavendustin A reduced the maximum current amplitudes to 18.7% ± 12.5% of the control (44.8% ± 17.9% recovery; n = 3; data not shown). It took lavendustin A 6 to 7 minutes to show a maximum effect. A recovery was observed 5 to 10 minutes after washout of genistein or lavendustin A. The inactive analogue of lavendustin A, lavendustin B (10 μM, Fig. 4 ) showed no effect on the maximum current amplitude (94.9% ± 2% of control in the presence of lavendustin B; n = 3). Herbimycin A (4 μM) took 10 to 15 minutes to have an effect. After 10 minutes, however, herbimycin A led to an irreversible reduction of the maximum current amplitude to 54.1% ± 20.9% of the control (after a 5 minute washout, 29.5% ± 20.6%; n = 3; data not shown). 
In the last set of control experiments, we tested the possibility that genistein acts only from the extracellular site of membrane. For this purpose, genistein (100 μM) was intracellularly applied through the patch pipette using the standard whole-cell configuration (Fig. 4C) . In these experiments the tip of the patch pipette contained control pipette solution, and the rest of the pipette was filled with genistein-containing pipette solution. Under these conditions the outwardly rectifying currents decreased in a few minutes. With genistein in the pipette solution, the maximum current amplitude after 4 minutes in the whole-cell configuration was 16.4% ± 7% of the current amplitude measured directly after breaking into the whole-cell configuration (n = 5). Without genistein in the pipette solution after 4 minutes in the whole-cell configuration, the maximum current amplitude was 99.3% ± 7.2% of the current amplitude measured directly after breaking into the whole-cell configuration (n =5; P = 0.0004). 
The results obtained by blocking tyrosine kinase were confirmed by experiments in which the tyrosine kinase pp60c-src was intracellularly applied through the patch pipette during the whole-cell configuration (Fig. 5) . For this purpose, the whole-cell configuration was established using a pipette solution containing MgATP (4 mM) and pp60c-src (30 U/mL). Maximum current amplitudes were then measured every minute after breaking into the whole-cell configuration and normalized to the first amplitude recorded. Control experiments (with either MgATP alone or MgATP and heat-inactivated pp60c-src in the pipette solution) disclosed a decline of delayed rectifier amplitudes over time in the whole-cell configuration. With active pp60c-src in the patch pipette, the maximum current amplitudes started to increase. This increase became statistically significant after 4 minutes in the whole-cell configuration and reached its maximum after 5 minutes. After 5 minutes in the whole-cell configuration, the delayed rectifier currents were 113% ± 17% of the control (n = 8) in control experiments (only ATP or heat-inactivated pp60c-src) and 422% ± 25% of the control (n = 4; P = 0.00,001) in experiments with active pp60c-src. At this time, in the whole-cell configuration, a current density of 2.8 ± 1.1 pA/pF was measured in control experiments and of 7.84 ± 1.5 pA/pF in experiments with active pp60c-src in the patch pipette (n = 5; P = 0.035). 
Western blot analysis of membrane proteins isolated from cultured RPE cells confirmed the expression of Kv1.3 α-subunits (Fig. 6) . Probing the blots with antibodies against Kv1.3 subunits revealed staining of a 67-kDa band that was not stained when antibodies were pretreated with the corresponding blocking peptides (Fig. 6A) . The same band was stained with antibodies against phosphorylated tyrosine residues (Fig. 6A) . When cells were pretreated with genistein (100 μM) before isolation of membrane proteins, the Western blot analysis stained only for the Kv1.3 subunits (Fig. 6B) but the Kv1.3 positive band was not stained by anti-phosphotyrosine antibodies (Fig. 6B)
Interaction of Tyrosine Kinase and Protein Kinase C in the Regulation of Delayed Rectifier K+ Channels
To obtain further information about the regulation of delayed rectifier currents the influence of protein kinase C (PKC) was studied. PKC was inhibited by chelerythrine (1 μM), which is most selective for PKC at the concentration used. 45 During application, the maximum current amplitude was 98.2% ± 7.5% of the control (109.2% ± 10.7% recovery; n = 6). A direct regulatory effect of PKC on the delayed rectifier channel was excluded in experiments in which PKC was activated by incubation of the cells in phorbol ester (Fig. 7) . For this purpose, the cells were incubated for 20 minutes in 1 μM phorbol-12-myristate-13-acetate (PMA) before electrophysiological recordings. They then showed delayed rectifier currents with a maximum amplitude of 3.8 ± 2 pA/pF (n = 4) and an activation time constant of 9.3 ± 1.2 ms (n = 4; analysis using the Hodgkin-Huxley type n 2 model). Neither of the values differed significantly from those in untreated cells. 
Application of genistein (50 μM) in the presence of the PKC blocker chelerythrine (1 μM) led to a reduction of the delayed rectifier currents comparable to that without PKC blocker. Under these conditions, genistein reduced the maximum current amplitude to 36% ± 5.2% of the control (95.5% ± 16% recovery; n = 6). Under these conditions genistein was slightly less effective, because we reduced the concentration of genistein in experiments in which two substances were applied at the same time. 
However, upregulation of PKC influenced the effect of tyrosine kinase on the delayed rectifier currents (Fig. 8) . For upregulation of PKC, cells were preincubated in phorbol ester (PMA, 1 μM) before electrophysiological recordings. In PMA-treated cells, inhibition of tyrosine kinase by genistein (50 μM) increased the maximum current amplitude to 159.5% ± 19.9% of the control (56% ± 15% recovery; n = 6). Application of daidzein (50 μM) to PMA-treated cells did not change the maximum current amplitude (104% ± 2.6% of control; recovery 115% ± 11% of control; n = 3 data not shown). These observations were confirmed by intracellular application of pp60c-src in PMA-treated RPE cells (Fig. 8B) . In cells pretreated by incubation in phorbol ester (1 μM PMA, for 20 minutes), intracellular application of pp60c-src (30 U/mL) led to significantly lower maximum current amplitudes after 8 minutes in the whole-cell configuration, compared with amplitudes in experiments with intracellular application of heat-inactivated pp60c-src. With active pp60c-src in the pipette, the maximum current amplitude after 8 minutes in the whole-cell configuration was 51% ± 15% (n = 5) of the amplitude measured directly after breaking into the whole-cell configuration. Under these conditions, the current density was 0.59 ± 0.07 pA/pF (n = 5) after 8 minutes in the whole-cell configuration. In control experiments using heat-inactivated pp60c-src (30 U/mL), the corresponding 8-minute values were 130% ± 30% (n = 4) of the current amplitude observed directly after breaking into the whole-cell configuration. With heat-inactivated pp60c-src, the current density was 2.31 ± 0.5 pA/pF (n = 4) after 8 minutes in the whole-cell configuration. 
Discussion
This study shows that tyrosine kinase can have opposite effects on the activity of Kv1.3 delayed rectifier potassium channels in RPE cells, depending on the activity level of PKC, which, by itself, has no regulatory effect on the potassium channel. However, tyrosine kinases of the src subtype are activators of Kv1.3 potassium channels in cells with unstimulated PKC but are inhibitors of that channel in cells with stimulated PKC. 
The depolarization-induced outwardly rectifying currents are potassium currents, as has been shown by various groups using pharmacologic and electrophysiologic evidence. 4 6 7 8 9 11 13 14 Immunocytochemical and PCR-based studies have demonstrated the expression of Kv1.2, Kv1.3, Kv1.4, Kv2.1, and Kv4.2 in the RPE. 15 The outwardly rectifying potassium currents in rat RPE cells displayed characteristics of delayed rectifier potassium channels with values for the activation threshold and the activation time constant comparable to those observed by Takahira and Hughes. 11 The delayed rectifier currents showed no fast inactivation, 14 which excludes the contribution of Kv1.4 and Kv4.2 α-subunits to the current. The currents were insensitive to hanatoxin, which is known to inhibit 80% of a current through Kv2.1 α-subunits at a concentration of 10−7 M. 46 Hanatoxin does not inhibit potassium channels composed of Kv1.3 subunits. 46 That Kv2.1 subunits do not contribute to the potassium currents in RPE cells is supported by the fact that the currents were blocked by agitoxin-2, which does not block currents through K+ channels composed of Kv2.1 subunits. 46 Furthermore, tityustoxin Kα did not influence delayed rectifier currents in RPE cells. Tityustoxin is known to block completely K+ channels with fast inactivation and those composed of Kv 1.2 subunits 47 at concentrations of 10−8 M. The delayed rectifier currents in RPE cells were completely blocked by agitoxin-2. At the concentration used, agitoxin completely blocks currents through K+ channels composed of Kv1.3 subunits. 48 Agitoxin-2 also has affinity with the Kv1.1 and Kv1.6 subunits, but the inhibition constants for blocking these subunits are 10 times higher than that for Kv1.3. The expression of Kv1.3 subunits was also confirmed in Western blot analysis of membrane proteins from RPE cells. In these blots the characteristic 67-kDa band was stained by antibodies against Kv1.3 subunits. The specificity of the staining was confirmed using the corresponding blocking peptides. Thus, the delayed rectifier currents are mostly currents through potassium channels composed of Kv1.3 α-subunits. Differences in the electrophysiological characteristics compared with heterologously expressed Kv1.3 channels may be due to the cellular environment in which the gene product is expressed. This has been reported, for example, for the Kv1.4 and Kv4.2 α-subunits, with different mammalian cell lines used as the expression system. 49  
Inhibition of protein tyrosine kinase by the three chemically different blockers genistein, lavendustin A, and herbimycin A 44 effectively reduced or fully blocked the depolarization-induced potassium currents. The inactive analogues daidzein (for genistein) and lavendustin B (for lavendustin A) showed no effects on delayed rectifier activity. Thus, the effects of tyrosine kinase blockers are probably due to inhibition of tyrosine kinase and not to unspecific binding to the potassium channel. Genistein is a blocker of several types of tyrosine kinase (e.g., inhibition constant for pp60c-src of 25 μM 50 ) and shows side effects on other serine-threonine protein kinases with inhibitory constants higher than 370 μM. 50 Lavendustin A is more specific for tyrosine kinase inhibition (e.g., inhibition constant for pp60c-src of 0.5 μM 51 ), but it shows effects on other protein kinases at concentrations higher than 100 μM. Herbimycin A blocks specific cytosolic tyrosine kinase of the src subtype (inhibition constant 10 μM 52 ) without affecting receptor tyrosine kinase or other protein kinases such as PKC or PKA. 52  
Furthermore, when genistein was intracellularly applied through the patch pipette during the whole-cell configuration we observed also an efficient inhibition of outwardly rectifying potassium channel activity. This points out that genistein probably acts through binding to tyrosine kinases in the cytosol and not by unspecific extracellular binding to the potassium channels. Thus, the delayed rectifier K+ channel seems to be activated by cytosolic tyrosine kinase of the src subtype. These results were confirmed by intracellular application of the tyrosine kinase pp60c-src, which increased the outwardly rectifying potassium current’s amplitude. In addition, we found that in Western blot analysis of membrane proteins from RPE cells, the band that was stained by antibodies against Kv1.3 subunits was also stained with antibodies against phosphorylated tyrosine side groups. The anti-phosphotyrosine staining was abolished when the cells were pretreated with genistein. This points to the involvement of tyrosine kinase in the tyrosine phosphorylation of Kv1.3 subunits. Thus, in resting RPE cells, tyrosine kinase of the pp60c-src subtype appears to be an activator of delayed rectifier potassium channels. 
PKC seemed not to have a direct effect on the delayed rectifier channels. The currents remained unchanged after treatment with PKC inhibitors or activators. In contrast, upregulation of PKC reversed the effect of tyrosine kinase on delayed rectifier potassium channels. This was shown either by use of tyrosine kinase inhibitors or by intracellular application of the pp60c-src kinase in cells pretreated with phorbol ester. The inactive analogues of the tyrosine kinase inhibitors showed no effect on the delayed rectifier currents. Thus, the effect of tyrosine kinase is dependent on PKC activity. Whereas pp60c-src activated outwardly rectifying potassium channels in cells with resting PKC activity, pp60c-src inhibited these ion channels in cells with stimulated PKC (Fig. 9) . Upregulation of PKC did not change the activity of delayed rectifier currents, PKC therefore does not directly influence the activity of the potassium channel or the activity of pp60c-src. We suggest that PKC determines the resultant effect on potassium channel activity only if tyrosine kinase activity is changed and not during constant tyrosine kinase activity. It is not clear how these two protein kinases interact at the channel protein. In addition, the involvement of other regulatory proteins cannot be excluded. Two tyrosine phosphorylation sites seem to be involved in the tyrosine kinase–dependent regulation of Kv1.3 subunits. 26 Analysis of the role of these two phosphorylation sites may help to explain how tyrosine kinase can have opposite effects on delayed rectifier activity. 
That pp60c-src can have opposite effects on delayed rectifier potassium channels in the same tissue depending on the activity of another protein kinase may partially explain contradictory observations in connection with the regulation of potassium channels by tyrosine kinase. We found in resting RPE cells that currents attributed to the Kv1.3 potassium channel α-subunit are stimulated by tyrosine kinase of the src subtype. This is an observation that is in contrast to investigations of Kv1.3 subunits in a heterologous expression system or in olfactory bulb neurons. 26 In addition, currents attributed to the Kv1.5 potassium channel α-subunit have been shown to be inhibited by tyrosine kinase of the src family when expressed in a mammalian cell line. 25 The currents of Kv1.5 α-subunits were found to be activated when investigated as native channels in Schwann cells. 27 Ca2+-activated potassium channels are also known to be activated by various types of tyrosine kinases but have also been found to be inhibited by tyrosine kinase. 30 32 35 36 37 38  
The role of the delayed rectifier potassium channels in the RPE is not understood. In many tissues, K+ channels are functional antagonists of voltage-dependent Ca2+ channels in the control of intracellular Ca2+ homeostasis. 21 We propose the same function for the delayed rectifier channel in RPE cells. In RPE cells, both voltage-dependent L-type Ca2+ channels and delayed rectifier K+ channels are activated by tyrosine kinase of the src subtype. 20 22 Thus, activation of tyrosine kinase led to simultaneous stimulation of both L-type channels and delayed rectifier channels. This limits the depolarization-induced activation of L-type channels and helps to control the influx of Ca2+ into the cell. In addition, the capability of controlling the activation of Ca2+ channels is dependent on the activity of PKC. Increased activation of Ca2+ channels with a subsequent increase in intracellular Ca2+ may require the enhanced activity of both tyrosine kinase and PKC. Under these conditions, the delayed rectifier K+ channels show reduced activity with increasing activity of tyrosine kinase. For example, this may be the case in the basic fibroblast growth factor–dependent stimulation of the Ca2+ second-messenger system, which involves the activation of L-type Ca2+ channels. 53  
In summary, we showed that delayed rectifier potassium currents can be attributed to Kv1.3 α-subunits. These potassium channels can be activated or inhibited by tyrosine kinase in the same tissue, depending on the activity of the PKC. To our knowledge, this is the first study that demonstrates tyrosine kinase interacting with other protein kinases to regulate the activity of delayed rectifier potassium channels. 
 
Figure 1.
 
Delayed outwardly rectifying currents in cultured RPE cells. (A) Protocol for electrical stimulation of RPE cells. The cell was depolarized by nine voltage steps of 10 mV increasing amplitude and 50 ms duration from a holding potential of 45 mV. This was followed by nine voltage steps of −10-mV increasing amplitude and 50-ms duration to hyperpolarize the cell. (B) Currents induced by the electrical stimulation shown in (A). (C) Plot of the maximum current amplitudes against the voltage steps of the electrical stimulation.
Figure 1.
 
Delayed outwardly rectifying currents in cultured RPE cells. (A) Protocol for electrical stimulation of RPE cells. The cell was depolarized by nine voltage steps of 10 mV increasing amplitude and 50 ms duration from a holding potential of 45 mV. This was followed by nine voltage steps of −10-mV increasing amplitude and 50-ms duration to hyperpolarize the cell. (B) Currents induced by the electrical stimulation shown in (A). (C) Plot of the maximum current amplitudes against the voltage steps of the electrical stimulation.
Figure 2.
 
Identification of the Kv-potassium channel subtype responsible for the outwardly rectifying current in RPE cells. (A) Extracellular application of charybdotoxin (10−7 M) led to a reduction of the outward current induced by a voltage step from −45 mV to +45 mV (stimulation shown at top). (B) Application of agitoxin-2 (10−8 M) nearly completely blocked the outward current. (C) Application of tityustoxin Kα (10−8 M) showed only weak effects on the outward current induced by voltage step from −45 mV to +45 mV. (D) Summary of all experiments performed to identify the subtype of Kv channel responsible for the outwardly rectifying current. Currents were plotted as a percentage of the currents observed before application of the blocker (data are the mean ± SEM; charybdotoxin: n = 4, P = 0.046; agitoxin: n = 4, P = 0.00,004; tityustoxin: n = 3, NS). **P < 0.01, ***P < 0.001.
Figure 2.
 
Identification of the Kv-potassium channel subtype responsible for the outwardly rectifying current in RPE cells. (A) Extracellular application of charybdotoxin (10−7 M) led to a reduction of the outward current induced by a voltage step from −45 mV to +45 mV (stimulation shown at top). (B) Application of agitoxin-2 (10−8 M) nearly completely blocked the outward current. (C) Application of tityustoxin Kα (10−8 M) showed only weak effects on the outward current induced by voltage step from −45 mV to +45 mV. (D) Summary of all experiments performed to identify the subtype of Kv channel responsible for the outwardly rectifying current. Currents were plotted as a percentage of the currents observed before application of the blocker (data are the mean ± SEM; charybdotoxin: n = 4, P = 0.046; agitoxin: n = 4, P = 0.00,004; tityustoxin: n = 3, NS). **P < 0.01, ***P < 0.001.
Figure 3.
 
Effect of various protein tyrosine kinase inhibitors on the outwardly rectifying current. (A) Currents induced by depolarizing part of the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of genistein (100 μM, right). (B) Currents induced by the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of herbimycin A (4 μM, right). (C) Summary of experiments to study the effect of tyrosine kinase inhibition. Maximum current amplitudes were normalized to the currents observed before application of the tyrosine kinase blocker (genistein: n = 5, P = 0.00041; lavendustin A (10 μM): n = 3, P = 0.013; herbimycin A: n = 3, P = 0.042).
Figure 3.
 
Effect of various protein tyrosine kinase inhibitors on the outwardly rectifying current. (A) Currents induced by depolarizing part of the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of genistein (100 μM, right). (B) Currents induced by the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of herbimycin A (4 μM, right). (C) Summary of experiments to study the effect of tyrosine kinase inhibition. Maximum current amplitudes were normalized to the currents observed before application of the tyrosine kinase blocker (genistein: n = 5, P = 0.00041; lavendustin A (10 μM): n = 3, P = 0.013; herbimycin A: n = 3, P = 0.042).
Figure 4.
 
Control experiments for the effects of tyrosine kinase inhibitors. Currents were activated by a voltage step from −45 mV to +45 mV for 50 ms. Currents under control conditions and (A) in the presence of 10 μM lavendustin B, the inactive analogue of lavendustin A; (B) in the presence of 100 μM daidzein, the inactive analogue of genistein; and (C) with intracellular application of 100 μM genistein through the patch pipette during the whole-cell configuration. For the experiments in (C) the patch-pipette was filled by a backfilling technique. The tip was filled with control pipette solution and the remainder of the patch pipette with pipette solution containing genistein. The figure shows the current just after and 4 minutes after breaking into the whole-cell configuration.
Figure 4.
 
Control experiments for the effects of tyrosine kinase inhibitors. Currents were activated by a voltage step from −45 mV to +45 mV for 50 ms. Currents under control conditions and (A) in the presence of 10 μM lavendustin B, the inactive analogue of lavendustin A; (B) in the presence of 100 μM daidzein, the inactive analogue of genistein; and (C) with intracellular application of 100 μM genistein through the patch pipette during the whole-cell configuration. For the experiments in (C) the patch-pipette was filled by a backfilling technique. The tip was filled with control pipette solution and the remainder of the patch pipette with pipette solution containing genistein. The figure shows the current just after and 4 minutes after breaking into the whole-cell configuration.
Figure 5.
 
Intracellular application of the tyrosine kinase pp60c-src with a patch pipette. Current induced by the depolarizing part of electrical stimulation shown in Figure 1A , (A) 2 minutes and (B) 8 minutes and after breaking into the whole-cell configuration with 4 mM MgATP and 30 U/mL pp60c-src in the patch pipette. (C) Normalized maximum current amplitudes were plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage step from −45 mV to +45 mV for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP (n = 4) or 4 mM MgATP and 30 U/mL pp60c-src, which was heat-inactivated by incubation at 95°C for 30 minutes (n = 4). The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly (P < 0.05) after 4 minutes in the whole-cell configuration. *P < 0.05, **P < 0.01.
Figure 5.
 
Intracellular application of the tyrosine kinase pp60c-src with a patch pipette. Current induced by the depolarizing part of electrical stimulation shown in Figure 1A , (A) 2 minutes and (B) 8 minutes and after breaking into the whole-cell configuration with 4 mM MgATP and 30 U/mL pp60c-src in the patch pipette. (C) Normalized maximum current amplitudes were plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage step from −45 mV to +45 mV for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP (n = 4) or 4 mM MgATP and 30 U/mL pp60c-src, which was heat-inactivated by incubation at 95°C for 30 minutes (n = 4). The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly (P < 0.05) after 4 minutes in the whole-cell configuration. *P < 0.05, **P < 0.01.
Figure 6.
 
Western blot analysis of Kv1.3 subunits in RPE cells. (A) Western blot of membrane proteins from cultured RPE cells in control conditions. The blot was stained with antibodies against Kv1.3 subunits (left), against Kv1.3 subunits pretreated with the corresponding blocking peptide (middle), and against phosphotyrosine (right). (B) Western blot of membrane proteins from cultured RPE cells that were incubated with genistein (100 μM). The blot was stained with antibodies against Kv1.3 subunits (left) and against phosphotyrosine (right). *P < 0.05.
Figure 6.
 
Western blot analysis of Kv1.3 subunits in RPE cells. (A) Western blot of membrane proteins from cultured RPE cells in control conditions. The blot was stained with antibodies against Kv1.3 subunits (left), against Kv1.3 subunits pretreated with the corresponding blocking peptide (middle), and against phosphotyrosine (right). (B) Western blot of membrane proteins from cultured RPE cells that were incubated with genistein (100 μM). The blot was stained with antibodies against Kv1.3 subunits (left) and against phosphotyrosine (right). *P < 0.05.
Figure 7.
 
Effect of PKC activation on delayed rectifier current. (A) Result of data analysis using the Hodgkin-Huxley n 2 model (smooth curve superimposed on the current record; current was induced by a voltage step from −45 mV to +45 mV). This model was used to calculate the activation time constant of delayed rectifying outward currents. (B) Effect of activating protein kinase by incubation in the phorbol ester PMA (1 μM for 20 minutes) on the activation time constant. The two values do not differ significantly. (C) Effect of activating protein kinase by incubation in phorbol ester on the current density of the delayed rectifying outward current. The two values do not differ significantly.
Figure 7.
 
Effect of PKC activation on delayed rectifier current. (A) Result of data analysis using the Hodgkin-Huxley n 2 model (smooth curve superimposed on the current record; current was induced by a voltage step from −45 mV to +45 mV). This model was used to calculate the activation time constant of delayed rectifying outward currents. (B) Effect of activating protein kinase by incubation in the phorbol ester PMA (1 μM for 20 minutes) on the activation time constant. The two values do not differ significantly. (C) Effect of activating protein kinase by incubation in phorbol ester on the current density of the delayed rectifying outward current. The two values do not differ significantly.
Figure 8.
 
Effect of tyrosine kinase on delayed rectifier potassium channels in cells with upregulated PKC. In these experiments the cells were incubated with PMA (1 μM) for 20 minutes before electrophysiological recordings. (A) Currents were induced by voltage step from −45 mV to +45 mV for 50 ms under control conditions after incubation in PMA and in the same cell in the presence of genistein (50 μM). (B) Intracellular application of pp60c-src in cells pretreated with phorbol ester. Normalized maximum current amplitudes plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage-step from −45 mV to +45 mv for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP and 30 U/mL pp60c-src, which was heat inactivated by incubation at 95°C for 30 minutes The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly after 8 minutes in the whole-cell configuration. *P < 0.05.
Figure 8.
 
Effect of tyrosine kinase on delayed rectifier potassium channels in cells with upregulated PKC. In these experiments the cells were incubated with PMA (1 μM) for 20 minutes before electrophysiological recordings. (A) Currents were induced by voltage step from −45 mV to +45 mV for 50 ms under control conditions after incubation in PMA and in the same cell in the presence of genistein (50 μM). (B) Intracellular application of pp60c-src in cells pretreated with phorbol ester. Normalized maximum current amplitudes plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage-step from −45 mV to +45 mv for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP and 30 U/mL pp60c-src, which was heat inactivated by incubation at 95°C for 30 minutes The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly after 8 minutes in the whole-cell configuration. *P < 0.05.
Figure 9.
 
Schematic drawing to summarize the data in the model. (A) Under control conditions, src subtype tyrosine kinase activated outwardly rectifying potassium channels composed of Kv1.3 subunits. (B) Stimulation of PKC did not influence the channel activity but changed the way in which tyrosine kinase regulated the channel activity. (C) When PKC was stimulated, tyrosine kinase of the src subtype inhibited the outwardly rectifying potassium channels.
Figure 9.
 
Schematic drawing to summarize the data in the model. (A) Under control conditions, src subtype tyrosine kinase activated outwardly rectifying potassium channels composed of Kv1.3 subunits. (B) Stimulation of PKC did not influence the channel activity but changed the way in which tyrosine kinase regulated the channel activity. (C) When PKC was stimulated, tyrosine kinase of the src subtype inhibited the outwardly rectifying potassium channels.
The authors thank Marianne Boxberger and Astrid Krolik for technical assistance, F. Stumpff for helpful discussions, and Kenton Swartz for kindly providing hanatoxin. 
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Figure 1.
 
Delayed outwardly rectifying currents in cultured RPE cells. (A) Protocol for electrical stimulation of RPE cells. The cell was depolarized by nine voltage steps of 10 mV increasing amplitude and 50 ms duration from a holding potential of 45 mV. This was followed by nine voltage steps of −10-mV increasing amplitude and 50-ms duration to hyperpolarize the cell. (B) Currents induced by the electrical stimulation shown in (A). (C) Plot of the maximum current amplitudes against the voltage steps of the electrical stimulation.
Figure 1.
 
Delayed outwardly rectifying currents in cultured RPE cells. (A) Protocol for electrical stimulation of RPE cells. The cell was depolarized by nine voltage steps of 10 mV increasing amplitude and 50 ms duration from a holding potential of 45 mV. This was followed by nine voltage steps of −10-mV increasing amplitude and 50-ms duration to hyperpolarize the cell. (B) Currents induced by the electrical stimulation shown in (A). (C) Plot of the maximum current amplitudes against the voltage steps of the electrical stimulation.
Figure 2.
 
Identification of the Kv-potassium channel subtype responsible for the outwardly rectifying current in RPE cells. (A) Extracellular application of charybdotoxin (10−7 M) led to a reduction of the outward current induced by a voltage step from −45 mV to +45 mV (stimulation shown at top). (B) Application of agitoxin-2 (10−8 M) nearly completely blocked the outward current. (C) Application of tityustoxin Kα (10−8 M) showed only weak effects on the outward current induced by voltage step from −45 mV to +45 mV. (D) Summary of all experiments performed to identify the subtype of Kv channel responsible for the outwardly rectifying current. Currents were plotted as a percentage of the currents observed before application of the blocker (data are the mean ± SEM; charybdotoxin: n = 4, P = 0.046; agitoxin: n = 4, P = 0.00,004; tityustoxin: n = 3, NS). **P < 0.01, ***P < 0.001.
Figure 2.
 
Identification of the Kv-potassium channel subtype responsible for the outwardly rectifying current in RPE cells. (A) Extracellular application of charybdotoxin (10−7 M) led to a reduction of the outward current induced by a voltage step from −45 mV to +45 mV (stimulation shown at top). (B) Application of agitoxin-2 (10−8 M) nearly completely blocked the outward current. (C) Application of tityustoxin Kα (10−8 M) showed only weak effects on the outward current induced by voltage step from −45 mV to +45 mV. (D) Summary of all experiments performed to identify the subtype of Kv channel responsible for the outwardly rectifying current. Currents were plotted as a percentage of the currents observed before application of the blocker (data are the mean ± SEM; charybdotoxin: n = 4, P = 0.046; agitoxin: n = 4, P = 0.00,004; tityustoxin: n = 3, NS). **P < 0.01, ***P < 0.001.
Figure 3.
 
Effect of various protein tyrosine kinase inhibitors on the outwardly rectifying current. (A) Currents induced by depolarizing part of the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of genistein (100 μM, right). (B) Currents induced by the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of herbimycin A (4 μM, right). (C) Summary of experiments to study the effect of tyrosine kinase inhibition. Maximum current amplitudes were normalized to the currents observed before application of the tyrosine kinase blocker (genistein: n = 5, P = 0.00041; lavendustin A (10 μM): n = 3, P = 0.013; herbimycin A: n = 3, P = 0.042).
Figure 3.
 
Effect of various protein tyrosine kinase inhibitors on the outwardly rectifying current. (A) Currents induced by depolarizing part of the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of genistein (100 μM, right). (B) Currents induced by the electrical stimulation shown in Figure 1A under control conditions (left) and currents of the same cell using the same electrical stimulation in the presence of herbimycin A (4 μM, right). (C) Summary of experiments to study the effect of tyrosine kinase inhibition. Maximum current amplitudes were normalized to the currents observed before application of the tyrosine kinase blocker (genistein: n = 5, P = 0.00041; lavendustin A (10 μM): n = 3, P = 0.013; herbimycin A: n = 3, P = 0.042).
Figure 4.
 
Control experiments for the effects of tyrosine kinase inhibitors. Currents were activated by a voltage step from −45 mV to +45 mV for 50 ms. Currents under control conditions and (A) in the presence of 10 μM lavendustin B, the inactive analogue of lavendustin A; (B) in the presence of 100 μM daidzein, the inactive analogue of genistein; and (C) with intracellular application of 100 μM genistein through the patch pipette during the whole-cell configuration. For the experiments in (C) the patch-pipette was filled by a backfilling technique. The tip was filled with control pipette solution and the remainder of the patch pipette with pipette solution containing genistein. The figure shows the current just after and 4 minutes after breaking into the whole-cell configuration.
Figure 4.
 
Control experiments for the effects of tyrosine kinase inhibitors. Currents were activated by a voltage step from −45 mV to +45 mV for 50 ms. Currents under control conditions and (A) in the presence of 10 μM lavendustin B, the inactive analogue of lavendustin A; (B) in the presence of 100 μM daidzein, the inactive analogue of genistein; and (C) with intracellular application of 100 μM genistein through the patch pipette during the whole-cell configuration. For the experiments in (C) the patch-pipette was filled by a backfilling technique. The tip was filled with control pipette solution and the remainder of the patch pipette with pipette solution containing genistein. The figure shows the current just after and 4 minutes after breaking into the whole-cell configuration.
Figure 5.
 
Intracellular application of the tyrosine kinase pp60c-src with a patch pipette. Current induced by the depolarizing part of electrical stimulation shown in Figure 1A , (A) 2 minutes and (B) 8 minutes and after breaking into the whole-cell configuration with 4 mM MgATP and 30 U/mL pp60c-src in the patch pipette. (C) Normalized maximum current amplitudes were plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage step from −45 mV to +45 mV for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP (n = 4) or 4 mM MgATP and 30 U/mL pp60c-src, which was heat-inactivated by incubation at 95°C for 30 minutes (n = 4). The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly (P < 0.05) after 4 minutes in the whole-cell configuration. *P < 0.05, **P < 0.01.
Figure 5.
 
Intracellular application of the tyrosine kinase pp60c-src with a patch pipette. Current induced by the depolarizing part of electrical stimulation shown in Figure 1A , (A) 2 minutes and (B) 8 minutes and after breaking into the whole-cell configuration with 4 mM MgATP and 30 U/mL pp60c-src in the patch pipette. (C) Normalized maximum current amplitudes were plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage step from −45 mV to +45 mV for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP (n = 4) or 4 mM MgATP and 30 U/mL pp60c-src, which was heat-inactivated by incubation at 95°C for 30 minutes (n = 4). The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly (P < 0.05) after 4 minutes in the whole-cell configuration. *P < 0.05, **P < 0.01.
Figure 6.
 
Western blot analysis of Kv1.3 subunits in RPE cells. (A) Western blot of membrane proteins from cultured RPE cells in control conditions. The blot was stained with antibodies against Kv1.3 subunits (left), against Kv1.3 subunits pretreated with the corresponding blocking peptide (middle), and against phosphotyrosine (right). (B) Western blot of membrane proteins from cultured RPE cells that were incubated with genistein (100 μM). The blot was stained with antibodies against Kv1.3 subunits (left) and against phosphotyrosine (right). *P < 0.05.
Figure 6.
 
Western blot analysis of Kv1.3 subunits in RPE cells. (A) Western blot of membrane proteins from cultured RPE cells in control conditions. The blot was stained with antibodies against Kv1.3 subunits (left), against Kv1.3 subunits pretreated with the corresponding blocking peptide (middle), and against phosphotyrosine (right). (B) Western blot of membrane proteins from cultured RPE cells that were incubated with genistein (100 μM). The blot was stained with antibodies against Kv1.3 subunits (left) and against phosphotyrosine (right). *P < 0.05.
Figure 7.
 
Effect of PKC activation on delayed rectifier current. (A) Result of data analysis using the Hodgkin-Huxley n 2 model (smooth curve superimposed on the current record; current was induced by a voltage step from −45 mV to +45 mV). This model was used to calculate the activation time constant of delayed rectifying outward currents. (B) Effect of activating protein kinase by incubation in the phorbol ester PMA (1 μM for 20 minutes) on the activation time constant. The two values do not differ significantly. (C) Effect of activating protein kinase by incubation in phorbol ester on the current density of the delayed rectifying outward current. The two values do not differ significantly.
Figure 7.
 
Effect of PKC activation on delayed rectifier current. (A) Result of data analysis using the Hodgkin-Huxley n 2 model (smooth curve superimposed on the current record; current was induced by a voltage step from −45 mV to +45 mV). This model was used to calculate the activation time constant of delayed rectifying outward currents. (B) Effect of activating protein kinase by incubation in the phorbol ester PMA (1 μM for 20 minutes) on the activation time constant. The two values do not differ significantly. (C) Effect of activating protein kinase by incubation in phorbol ester on the current density of the delayed rectifying outward current. The two values do not differ significantly.
Figure 8.
 
Effect of tyrosine kinase on delayed rectifier potassium channels in cells with upregulated PKC. In these experiments the cells were incubated with PMA (1 μM) for 20 minutes before electrophysiological recordings. (A) Currents were induced by voltage step from −45 mV to +45 mV for 50 ms under control conditions after incubation in PMA and in the same cell in the presence of genistein (50 μM). (B) Intracellular application of pp60c-src in cells pretreated with phorbol ester. Normalized maximum current amplitudes plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage-step from −45 mV to +45 mv for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP and 30 U/mL pp60c-src, which was heat inactivated by incubation at 95°C for 30 minutes The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly after 8 minutes in the whole-cell configuration. *P < 0.05.
Figure 8.
 
Effect of tyrosine kinase on delayed rectifier potassium channels in cells with upregulated PKC. In these experiments the cells were incubated with PMA (1 μM) for 20 minutes before electrophysiological recordings. (A) Currents were induced by voltage step from −45 mV to +45 mV for 50 ms under control conditions after incubation in PMA and in the same cell in the presence of genistein (50 μM). (B) Intracellular application of pp60c-src in cells pretreated with phorbol ester. Normalized maximum current amplitudes plotted against time in the whole-cell configuration. Maximum current amplitudes were measured for a voltage-step from −45 mV to +45 mv for 50 ms every 1 minute in the whole-cell configuration and normalized to the current amplitude measured directly after breaking into the whole-cell configuration. The control experiments (n = 8) were performed with a pipette solution containing 4 mM MgATP and 30 U/mL pp60c-src, which was heat inactivated by incubation at 95°C for 30 minutes The pp60c-src experiments (n = 5) were performed with active tyrosine kinase in the pipette solution. Data differ significantly after 8 minutes in the whole-cell configuration. *P < 0.05.
Figure 9.
 
Schematic drawing to summarize the data in the model. (A) Under control conditions, src subtype tyrosine kinase activated outwardly rectifying potassium channels composed of Kv1.3 subunits. (B) Stimulation of PKC did not influence the channel activity but changed the way in which tyrosine kinase regulated the channel activity. (C) When PKC was stimulated, tyrosine kinase of the src subtype inhibited the outwardly rectifying potassium channels.
Figure 9.
 
Schematic drawing to summarize the data in the model. (A) Under control conditions, src subtype tyrosine kinase activated outwardly rectifying potassium channels composed of Kv1.3 subunits. (B) Stimulation of PKC did not influence the channel activity but changed the way in which tyrosine kinase regulated the channel activity. (C) When PKC was stimulated, tyrosine kinase of the src subtype inhibited the outwardly rectifying potassium channels.
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