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Endocrinology Vol. 139, No. 1 179-188
Copyright © 1998 by The Endocrine Society

ARTICLES

Activation of Calcium-Permeable Cation Channel by Insulin in Chinese Hamster Ovary Cells Expressing Human Insulin Receptors1

Lin Nie, Makoto Kanzaki, Hiroshi Shibata and Itaru Kojima

Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan

Address all correspondence and requests for reprints to: Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan. E-mail: ikojima{at}news.sb.gunma-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was conducted to examine the ability of insulin receptor to activate the calcium signaling system in Chinese hamster ovary (CHO) cells expressing human insulin receptor (CHO-IR cells). In these cells, insulin evoked the elevation of cytoplasmic free calcium concentration, [Ca2+]c, measured by using fura-2. Insulin-induced increase in [Ca2+]c was blocked by reducing the extracellular calcium concentration to 1 µM or by adding nickel chloride, an inorganic inhibitor of calcium entry. Insulin did not elevate [Ca2+]c in parental CHO cells or in CHO cells expressing mutant insulin receptor lacking an ATP-binding site. When the transmembrane calcium current was measured by perforated whole-cell patch clamp, adding insulin to the bath solution markedly augmented the inward calcium current. In a cell-attached patch, a single channel activity appeared when insulin was included in the pipette. In contrast, insulin added outside the patch was ineffective. The current/voltage relationship demonstrated that insulin activated a voltage-independent calcium-permeable cation channel with a single-channel conductance of 10 pS. Exposing CHO-IR cells to pertussis toxin abolished the subsequent insulin effect on [Ca2+]c and activation of the calcium-permeable channel. Mastoparan activated the 10-pS calcium-permeable cation channel. In an inside-out patch, insulin activated the calcium-permeable channel when the bath solution contained both GTP and ATP. Nonhydrolyzable ATP could substitute for ATP. These results indicate that in CHO-IR cells, insulin elevates [Ca2+]c by activating the 10-pS calcium-permeable cation channel. Activation by the insulin receptor involves pertussis toxin-sensitive G protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN induces pleiotropic effects in various types of target cells (1, 2, 3). Insulin is the principal hormone that controls blood glucose levels: it augments the transport of glucose into muscle cells and adipocytes and modulates glycogen metabolism in the liver. Additionally, insulin alters the activity and expression of many enzymes in various types of cells. The action of insulin is exerted through the insulin receptor, a transmembrane protein with intrinsic protein kinase activity (1, 2, 3). Binding of insulin to the {alpha}-subunit of the insulin receptor results in the activation of tyrosine kinase located in the ß-subunit. This leads to the autophosphorylation of the receptor and other substrates, including insulin receptor substrate-1 (IRS-1). Intracellular signaling pathways, activated by the insulin receptor, are diverse; and it is considered that intrinsic protein tyrosine kinase is critical for the signal transduction of insulin actions. The pleiotropic action of insulin is mediated by diverse signaling pathways that separate at the insulin receptor (3). It has been postulated that calcium is involved in some of the actions of insulin (4, 5, 6), but this remains controversial. In an early study, Draznin et al. (7) reported that insulin increases the cytoplasmic free calcium concentration, [Ca2+]c, in adipocytes, whereas others showed that insulin has no effect on [Ca2+]c (8, 9). Recent studies, using improved methods, show that insulin affects cellular calcium metabolism (10, 11, 12, 13). In addition, molecules involved in cellular calcium signaling are modified by insulin. For example, calmodulin is phosphorylated by the insulin receptor (14, 15). Nevertheless, whether insulin can activate the calcium signaling system remains to be established. In this regard, receptor for insulin-like growth factor-I (IGF-I), which has a structure similar to that of the insulin receptor, can activate the calcium messenger system. In Balb/c 3T3 fibroblasts, IGF-I increases the cytoplasmic free calcium concentration ([Ca2+]c) by activating calcium-permeable channels (16, 17). Similarly, IGF-I stimulates calcium entry in Chinese hamster ovary (CHO) (18), FRTL thyroid (19), and renal tubular cells (20). Given the structural similarity, the insulin receptor also may activate the calcium signaling system. Therefore, we studied the effect of insulin on cellular calcium metabolism in CHO cells expressing either human insulin receptor (CHO-IR) or mutant insulin receptor (mIR) lacking ATP-binding site (CHO-mIR1030). The results indicated that in CHO-IR cells, insulin activates calcium-permeable channels and thereby increases [Ca2+]c.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Wild-type CHO cells were obtained from The Riken Cell Bank (Tsukuba, Japan). CHO-IR cells (21) were generously provided by Dr. T. Kadowaki of the University of Tokyo (Tokyo, Japan). CHO cells expressing the mIRs lacking ATP-binding site (CHO-mIR1030) or mIRs in which tyrosine-972 was replaced with phenylalanine (CHO-mIR972) were established by stably transfecting CHO cells with mutant IR-complementary DNA (cDNA). All cell lines were routinely maintained in Ham’s F12 medium supplemented with 2 mM glutamine, 150 mg/ml penicillin, 100 mg/ml streptomycin, and 10% FBS (GIBCO, Grand Island, NY) at 37 C, in a humidified incubator containing 95% air and 5% CO2.

Measurement of cytoplasmic free calcium concentration
Cytoplasmic free calcium concentration ([Ca2+]c) in a single cell was measured using the fluorescent probe fura-2 (22). Fura-2 acetoxymethyl ester (fura-2/AM) was dissolved in dimethyl sulfoxide (DMSO) and stored at -20 C. The cells were grown on glass coverslips. Twenty-four hours later, cells in the growth phase were rinsed twice with normal extracellular solution consisting of 137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 10 mM HEPES/NaOH (pH 7.4). When the extracellular calcium concentration was reduced to 1 µM, Ca2+-EGTA buffer was used (Ca/EGTA = 0.97) (16). Cells in normal extracellular solution were loaded with 2 ml of 1 µM fura-2/AM and incubated for 30 min at room temperature. Cells were washed twice with the loading buffer, and dyes were deesterified for 20 min. The coverslips were then placed in a flow-through chamber mounted on the stage of a TMD microscope (Nikon, Tokyo, Japan). Fluorescence records were taken at excitation {lambda} of 340 nm and 380 nm and emission of 510 nm. The 340/380 ratios were recorded using an Argus 100 (Hamamatsu Photonics, Hamamatsu, Japan), and the signal was calibrated in terms of cytoplasmic free calcium concentration, as described previously (23). Most of the experiments were carried out at 28–34 C. In each experiment, the 340/380 ratio was monitored in 10–20 cells, and at least 500 cells were examined for each condition to determine the response to insulin.

Whole-cell patch clamp analysis
To evaluate the Ca2+ influx induced by insulin in CHO-IR cells, we used the pore-forming antibiotic amphotericin B to produce perforated whole-cell patches. The criteria for selection of a cell for electrical measurement were that the cell had no contact with neighboring cells and had a minimum of extracellular debris. A stock solution of amphotericin B (60 mg/ml) (WAKO, Osaka, Japan) was prepared once per week, by dissolving in DMSO, and stored at -20 C. This stock solution was added to the pipette solution at a concentration of 240 mg/ml. Amphotericin B-perforated patches were obtained as described by Horn and Matoy (24). Pipettes were filled with the amphotericin B solution and gently achieved a G{Omega} (>2 G{Omega}) seal between the tip of a fire-polished patch pipette and the cell membrane. Typically, after 5–10 min exposure to amphotericin B, maximal whole-cell currents were recorded using a computer-based amplifier system and a List EPC9 patch clamp amplifier (List, Darmstadt, Germany) controlled by E9 screen software (HEKA, Lambrecht, Germany). Voltage ramps were of 300 msec duration and ranged from a holding potential at -100 to +100 mV. Capacitance and series resistance were canceled by the automatic neutralization routine of the EPC-9. The pipette solution contained (in mM): CsOH, 142.5; aspartate, 142.5; HEPES (pH 7.1), 10; sucrose, 20; EGTA-Cs, 1; MgSO4, 4; and amphotericin, B 0.2. The bath solution contained (in mM): Ca(OH)2, 10; N-methyl-D-glucamine, 140; methanesulfonic acid, 140; and HEPES (pH 7.4), 10. Under these conditions, the principal membrane permeant ion was Ca2+.

Single-channel recordings
Single-channel recordings were made in the cell-attached mode. After fire-polishing, the electrode resistance was about 8 M{Omega}. A typical patch had a seal resistance greater than 20 G{Omega} brought about by gentle suction. Data were collected after filtering at 100 Hz and analyzed by using the TAC program (HEKA). The pipette solution comprised 110 mM BaCl2 and 10 mM HEPES (pH 7.4). Tetrodotoxin was added to the pipette solution at a final concentration of 200 nM to block any voltage-dependent Na current. The bath solution comprised (in mM): NaCl, 137; KCl, 5; MgCl2, 1; HEPES (pH 7.4), 10; CaCl2, 2; and glucose, 5. All experiments were performed at room temperature.

DNA transfection
The cDNA encoding the human insulin receptor with a mutation (Lys-Met) at lysine 1030 (25) and a mutant (Tyr-Phe) at tyrosine 972 (26) in the pcDL-SR296 eukaryotic expression vector were provided by Dr. Y. Ebina of Tokushima University (Tokushima, Japan). The CHO cells were transfected by using DOTAP, according to the manufacturer’s recommendation. CHO cells were seeded at a density of 5 x 105/100-mm plastic culture dish and cultured for 20 h before transfection. Cells were cotransfected with PSV2-neo, a selectable marker conferring neo resistance. Seven micrograms of SR-mIR cDNA and 0.5 µg PSV2-neo (relative mass ratio of 15:1) were suspended on 75 µl DOTAP/HEPES solution and incubated at room temperature for 10–15 min. Before transfection, the medium of CHO cells was replaced with FCS-free F-12 medium, and the DOTAP/DNA mixture was added to the media. After a 3-h incubation at 37 C, FCS was added to the medium to a final concentration of 10%. Ten hours after transfection, the medium containing DOTAP/DNA was replaced with fresh medium and incubated for another 36 h. The cells were then trypsinized and replated at a density of 3 x 105 cells/100-mm dish in selection medium containing 800 mg/ml of the neomycin analog G418. After 10–14 days, G418-resistant single colonies were selected and screened for high levels of expression of mIR by determining insulin binding.

Measurement of insulin binding
Cells were incubated for 10 h at 12 C in 0.5 ml binding buffer [100 mM HEPES (pH 8.0), 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 8 mM glucose, and 10 mg/ml BSA] containing [125I]insulin (200 pM, 40,000 cpm) in the presence and absence of 2 µM unlabeled insulin. The unbound insulin was removed from the monolayers by washing three times with ice-cold binding buffer. The cells were solubilized with 1 ml of 0.5 M NaOH, and the bound radioactivity was measured by a {gamma}-counter. For measurement of the number of binding sites, cells were incubated with various concentrations of labeled insulin, and the number of specific insulin-binding sites was obtained by Scatchard analysis.

Affinity labeling of the insulin receptor
Confluent cells, cultured in 100-mm dishes, were incubated with 3 ml of binding buffer containing 3 nM [125I]-insulin (6.2 x 105 cpm) for 10 h at 12 C. Unbound insulin was removed by washing the cells twice with ice-cold PBS. Freshly prepared disuccinimidyl suberate, dissolved in DMSO, was added to a final concentration of 0.5 mM, and the cells were incubated at 4 C for 15 min. The reaction was terminated by an addition of 1 ml of 20 mM Tris/HCl (pH 7.4) buffer containing 150 mM NaCl and 1 mM EDTA. The cells were scraped into a 1.5-ml Eppendorf tube, centrifuged at 2,000 rpm for 5 min at 4 C, and solubilized by adding 50 ml lysis buffer containing 1% Triton X-100. Nuclei and detergent-insoluble materials were removed by centrifugation at 15,000 rpm for 15 min at 4 C. Fifty microliters of 2 x Laemmli sample buffer were added to the supernatant, and the samples were boiled for 3 min. The proteins were separated by the reducing SDS-PAGE on 7.5% gel and visualized by a BAS-2000 Imaging Analyzer (Fuji Film, Tokyo, Japan). The relative molecular weights of the affinity-labeled proteins were determined using rainbow-colored protein weight markers.

Analysis of tyrosine phosphorylation induced by insulin
Confluent cells, cultured in 100-mm dishes, were serum starved for 24 h. The cells were incubated in phosphate-free DMEM for 1 h, and then in phosphate-free DMEM containing 0.4 mCi/ml [32P]orthophosphate for 3 h.

The cells were incubated for 5 min with 100 nM insulin, washed with ice-cold Tris-buffered saline three times, and lysed in 0.8 ml lysis buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM sodium orthovanadate, 2 mM phenylmethylsolfonyl fluoride, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 40 mg/ml aprotinin, 4 mM EDTA, 5% glycerol, and 1% Triton X-100. The cells were scraped into a 1.5-ml Eppendorf tube, and the insoluble materials were removed by centrifugation at 15,000 rpm for 20 min at 4 C. The supernatant was immunoprecipitated with monoclonal antiphosphotyrosine antibody ({alpha}PY). The cell extracts (0.5 ml) were incubated with 2 µg {alpha}PY for 2 h at 4 C with constant shaking. The antigen-antibody complex was precipitated by incubation with 30 µl protein G-Sepharose for 1 h at 4 C, and immunoprecipitates were washed three times with washing buffer A containing 50 mM HEPES (pH 7.4), 500 mM NaCl, 0.1% Triton X-100, 0.005% SDS, 100 mM sodium fluoride, 2 mM sodium orthovanadate, 1 mM dithiothreitol, and once with washing buffer B containing 10 mM Tris/HCl (pH 7.5), 100 mM NaCl, 2 mM sodium orthovanadate, and 1 mM dithiothreitol. The proteins were eluted from the pellet by boiling 3 min in 2 x Laemmli sample buffer and separated by reducing SDS-PAGE. The phosphoproteins were identified with a BAS-2000 Imaging Analyzer. For measurement of IRS-1 phosphorylation, cells were incubated for 5 min with 100 nM insulin. The cells were lysed and immunoprecipitated with anti-IRS-1 antibody. The samples were electrophoresed on SDS-PAGE, transferred to nylon membrane, blocked in 3% BSA-rinse buffer [10 mM Tris/HCl (pH 7.5), 150 mM NaCl, EDTA, and 0.05% Tween 20] at room temperature for 3 h, and blotted with monoclonal {alpha}PY (final concentration of 0.5 mg/ml) overnight at 4 C with constant shaking. After washing the membrane with rinse buffer 3 times, the bound antibody was detected by incubation with 0.1 mCi/ml of [125I]-protein A for 1 h at room temperature, followed by autoradiography. The activity of phosphatidylinositol 3-kinase (PI 3-kinase), MAP kinase, and the GTP-bound form of Ras was measured as described elsewhere (27).

Measurement of DNA synthesis
DNA synthesis was assessed by measuring the incorporation of [3H]thymidine into tricholoroacetic acid-precipitable materials. CHO-IR cells were seeded in a 24-well plate at a density of 1 x 104/well. Twenty-four hours later, the medium was changed, and the cells were incubated for 72 h with serum-free medium. The cells were then incubated for 16 h in medium containing 1 mCi/ml [3H]thymidine in the presence or absence of insulin, and [3H]thymidine incorporation was measured as described (16).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the cells
We characterized the cells used in this study by measuring the number of IR, affinity cross-linking of IR, and tyrosine phosphorylation of cellular proteins in response to insulin. As shown in Table 1Go, large numbers of IR were detected in CHO cells expressing either IR or mutant IR, whereas the number of IR was much less in parental CHO cells. Note that the number of IR in CHO-mIR cells was less than in CHO-IR cells but was comparable with that in previous studies (25). Figure 1AGo shows the affinity cross-linking of CHO, CHO-IR, and CHO-mIR1030 cells with [125I]insulin. A 130-KDa protein (presumably, the {alpha}-subunit of the insulin receptor) was detected in CHO-IR and CHO-mIR1030 cells, which disappeared in the presence of excess unlabeled insulin (data not shown). Figure 1BGo demonstrates the tyrosine-phosphorylated proteins in CHO, CHO-IR, and CHO-mIR1030 cells incubated with insulin. Tyrosine-phosphorylated proteins with molecular masses of 180 KDa, 130 KDa, 95 KDa, and 56 KDa, were detected in CHO-IR but not in either CHO or CHO-mIR1030 cells. The 180-KDa and 95-KDa proteins were presumably IRS-1 and the ß-subunit of IR, respectively. mIR972 lacks the tyrosine phosphorylation site (tyrosine-972) to which IRS proteins and Shc bind. Phosphorylation of these proteins were impaired, although the receptor kinase activity remained intact (26). Insulin induced tyrosine phosphorylation of IRS-1 in CHO-IR cells, whereas IRS-1 was not phosphorylated by insulin in CHO-mIR972 cells (Fig. 1CGo).


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  		Table 1. Numbers of insulin-binding sites in CHO, CHO-IR, and CHO-mIR cells

 


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  		Figure 1. Characterization of the cells used in this study. Panel A, Affinity cross-linking was done using [125I]insulin and disuccinimidyl suberate in CHO, CHO-IR, and CHO-mIR1030 cells; panel B, CHO, CHO-IR, and CHO-mIR1030 cells, labeled with [32P], were incubated for 5 min with 100 nM insulin. The cell lysates were immunoprecipitated with {alpha}PY. The precipitates were subjected to SDS-PAGE, followed by autoradiography. Panel C, CHO, CHO-IR, and CHO-mIR972 cells were incubated for 5 min with 100 nM insulin. Cells were lysed, and IRS-1 was immunoprecipitated. Phosphorylation of IRS-1 was assessed by Western blotting using {alpha}PY, as described in Materials and Methods.

 
Effect of insulin on [Ca2+]c
In CHO-IR cells, 10 nM insulin elevated [Ca2+]c. The [Ca2+]c response to insulin was variable, and [Ca2+]c was elevated in approximately 75% of the cells in the growth phase (582 of 780 cells). However, when CHO-IR cells were growth-arrested by incubating in serum-free medium for 2 days, there was no apparent [Ca2+]c response to insulin (none of 560 cells; data not shown). When cells in growth phase were incubated with IGF-I (instead of serum) for 2 days, the effect of insulin on [Ca2+]c was observed (data not shown). Figure 2AGo shows the typical, rapid increase in [Ca2+]c found in most insulin-treated cells. In some cells, [Ca2+]c was elevated after a lag period of 20–50 sec (54 of 780 cells). Elevation of [Ca2+]c lasted for several minutes, and it frequently oscillated. The [Ca2+]c response to insulin was totally dependent on extracellular calcium, and insulin did not elevate [Ca2+]c in medium containing 1 µM calcium (none of 620 cells) (Fig. 2BGo). Similarly, insulin did not increase [Ca2+]c in cells incubated in medium containing 0.25 mM NiCl2 or LaCl3 (data not shown). Insulin did not induce the elevation of [Ca2+]c in parental CHO (Fig. 2CGo) or CHO-mIR1030 cells (Fig. 2DGo). When CHO-IR cells were stimulated by 1 nM insulin, [Ca2+]c was elevated in approximately 10% of the cells (82 cells of 800 cells) (Fig. 3AGo). However, when cells were first incubated in calcium-free buffer, stimulated with 1 nM insulin, and the extracellular calcium concentration raised to 2 mM in the presence of insulin, [Ca2+]c was elevated in 75% of the cells (463 of 620 cells) (Fig. 3BGo). Note that [Ca2+]c did not change significantly after the change of extracellular calcium when 1 nM insulin was absent. These results are consistent with the notion that 1 nM insulin augmented calcium entry.



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  		Figure 2. Effect of insulin on [Ca2+]c in CHO, CHO-IR, and CHO- mIR1030 cells. Fura-2-loaded CHO-IR cells were stimulated by 10 nM insulin in the presence of 2 mM (A) or 1 µM (B) extracellular calcium. Fura-2 fluorescence in a single cell was monitored. Fura-2-loaded CHO (C) or CHO-mIR1030 (D) cells were incubated with 10 nM insulin in the presence of 2 mM extracellular calcium, and the fura-2 fluorescence was monitored.

 


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  		Figure 3. Effect of insulin on [Ca2+]c in CHO-IR cells. A, Fura-2-loaded CHO-IR cells were stimulated by 1 nM insulin, and fura-2 fluorescence in a single cell was monitored; B, Fura-2-loaded CHO-IR cells were incubated in medium containing 1 µM calcium, then 1 nM insulin (•) or saline ({circ}) was added. Extracellular calcium concentration was then raised to 2 mM, as indicated. Fura-2 fluorescence in a single cell was monitored.

 
Effect of insulin on calcium current in CHO-IR cells
To further examine the effect of insulin on calcium entry, we measured the transmembrane calcium current using a whole-cell patch clamp and amphotericin B (24). The extracellular and intracellular solutions contained only calcium as the permeant ion. Under these conditions, calcium is the only ion that passes across the plasma membrane. In unstimulated cells, the transmembrane calcium current was small when voltage ramps between -100 mV and +100 mV were applied. When 10 nM insulin was added to the same cell, the application of voltage ramps induced an inward current carried by Ca2+ (189 of 246 cells) (Fig. 4AGo). The current-voltage (I/V) relationship was linear, suggesting that Ca2+ entered the cells via a voltage-independent pathway. The insulin-mediated inward Ca2+ current was not inhibited by nifedipine, a blocker of L-type voltage-dependent channels, but was completely blocked by 0.25 mM NiCl2 or LaCl3 (data not shown). Figure 4BGo shows the dose-response relationship for the insulin-induced inward current. The effect of insulin was detected at 5 x 10-10 M. Figure 5AGo shows a single-channel recording of the insulin-activated calcium-permeable channel, measured by using Ba2+ as a charge carrier. Currents were recorded using a cell-attached patch with 1 nM insulin in the pipette. Calcium-permeable channel activity was detected when insulin was in the pipette (92 of 100 patches); but in the absence of insulin, the activity was barely detected (none of 70 patches) (Fig. 5BGo). Insulin added outside the patch did not activate the channel, suggesting that the channel is regulated tightly by the insulin receptor. Figure 5CGo shows the I/V relationship of the insulin-activated calcium-permeable channel. The I/V relationship was linear, and the unitary conductance was 10 pS. Note that the channel with the same single-channel conductance was activated when 1 nM IGF-I was included in the pipette (data not shown). Hence, the same channel also may be activated by the IGF-I receptor. The channel activity was not detected in parental CHO or CHO-mIR1030 cells with insulin in the pipette (data not shown). In contrast, the channel activity was detected in CHO-mIR972 cells. As shown in Fig. 6Go, insulin added inside the pipette activated the channel (35 of 36 patches).



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  		Figure 4. Transmembrane calcium current induced by insulin. A, A CHO-IR cell was incubated in a bath solution containing calcium, as described in Materials and Methods. Transmembrane calcium current was measured using a perforated whole cell patch clamp. A voltage ramp from -100 mV to +100 mV was applied in the same cell in the presence or absence of 10 nM insulin in the bath solution. B, Inward calcium current was measured in the presence of various concentrations of insulin, as described above, and the net inward current obtained at -100 mV was plotted as a function of insulin concentration. Values are the means ± SE for four experiments.

 


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  		Figure 5. Single-channel current induced by insulin. Single-channel current was recorded in a cell-attached patch using Ba2+ as a charge carrier. Insulin (1 nM) was added inside (A) or outside (B) the patch. C, I/V relationship for insulin-activated Ba2+ current measured in the cell-attached patch.

 


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  		Figure 6. Effect of insulin on single-channel activity in CHO-mIR972 cells. Single-channel current was measured in CHO-mIR972 cells, using a cell-attached patch, in the presence of 1 nM insulin in the pipette.

 
Effect of pertussis toxin (PTX) on insulin-induced elevation of [Ca2+]c
We examined whether PTX affects the insulin-induced elevation of [Ca2+]c in CHO-IR cells. Cells were pretreated for 4 h with 100 ng/ml PTX, and the effect of insulin was examined. As shown in Fig. 7Go, 10Go nM insulin did not induce elevation of [Ca2+]c in PTX-treated cells (none of 610 cells). Similarly, calcium-permeable channel activity was not detected in a cell-attached patch in PTX-treated cells (none of 90 patches; data not shown). To determine the involvement of PTX-sensitive G protein in the activation of insulin-operated channel, we tested the effect of mastoparan, an activator of PTX-sensitive G proteins (28), in the cell-attached patch. As shown in Fig. 8AGo, calcium-permeable channels were markedly activated by mastoparan (20 of 20 patches), whereas mas-17, an inactive analog of mastoparan, was ineffective (none of 25 patches) (Fig. 8BGo).



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  		Figure 7. Effect of pretreatment with PTX. CHO-IR cells were incubated for 4 h with 100 nM PTX, and then the effect of insulin on [Ca2+]c was measured.

 


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  		Figure 10. Effect of nickel chloride on insulin-induced DNA synthesis. CHO-IR cells were incubated for 16 h with or without 10 nM insulin in the presence or absence of 0.25 mM NiCl2, then [3H]thymidine incorporation was measured. Values are the means ± SE for four experiments. *, P < 0.05 vs. without NiCl2. Statistical significance was evaluated by Student’s t test.

 


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  		Figure 8. Effect of mastoparan on the 10-pS calcium-permeable channel. A single-channel barium current was measured in a cell-attached patch containing 100 nM mastoparan (A) or Mas-17 (B).

 
Effect of GTP and ATP on insulin-induced activation of calcium-permeable channel
To further determine the role of G protein in insulin-induced activation of the calcium-permeable cation channel, we studied the effect of nucleotides on insulin-activated Ba2+ current in excised patches. We first recorded the channel in cell-attached mode with insulin in the pipette (64 patches) (Fig. 9AGo). The inside-out patch was then obtained after confirming channel activity in the patch. The channel activity disappeared in all patches tested. In the presence of 2 mM MgCl2 alone, channel activity was undetectable, even in the presence of insulin in the pipette (none of 64 patches) (Fig. 9BGo). When 1 mM ATP, 1 mM GTP, and 2 mM MgCl2 were included in the bath solution, the channel activity became detectable (64 of 64 patches) (Fig. 9CGo). Removing ATP (Fig. 9DGo) or GTP (data not shown) abolished the channel activity. The nonhydrolyzable ATP, AMP-PNP, could substitute for ATP. Thus, channels were active in the presence of GTP, MgCl2, and AMP-PNP (64 of 64 patches) (Fig. 9EGo). Addition of 1 mM GDP-ßS blocked activation of the channel in the presence of ATP, GTP, and MgCl2 (data not shown). Anti-Ras antibody did not affect the activation of the channel in the presence of ATP, GTP, and MgCl2 (data not shown). In the absence of insulin in the pipette, no channel activity was detected in the excised mode (even in the presence of ATP, GTP, and magnesium; data not shown). When mastoparan was included in the patch pipette, 10-pS channel was activated in the excised patch with 1 mM GTP and 2 mM MgCl2 in the bath (data not shown). ATP was not required for channel activation by mastoparan.



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  		Figure 9. Effect of nucleotides on insulin-activated channel. The 10-pS calcium-permeable channel was recorded in a cell-attached patch with 1 nM insulin in the pipette (A). An excised patch was obtained, and the channel activity was recorded. The bath solution contained 2 mM MgCl2 (B); 1 mM GTP, 2 mM MgCl2, and 1 mM ATP (C); 2 mM MgCl2 and 1 mM GTP (D); and 1 mM GTP, 2 mM MgCl2, and 1 mM AMP-PNP (E). The results are representative of 64 experiments with similar results.

 
Effect of blockade of calcium entry on insulin-induced DNA synthesis
To understand the biological significance of insulin- activated calcium-permeable channels, we examined the effect of NiCl2 on insulin-induced DNA synthesis in CHO-IR cells. As shown in Fig. 10Go, 0.25 mM NiCl2, which inhibits insulin-induced calcium entry, blocked insulin-induced DNA synthesis, as assessed by [3H]thymidine incorporation. Note that NiCl2 did not affect insulin-induced activation of Ras (Fig. 11AGo), MAP kinase (Fig. 11BGo), or PI 3-kinase (Fig. 11CGo).



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  		Figure 11. Effect of nickel chloride on insulin-induced activation of Ras, MAP kinase, and PI 3-kinase. CHO-IR cells were incubated for 5 min with 100 nM insulin in the presence and absence of 0.25 mM NiCl2. Activities of Ras (A), MAP kinase (B), and PI 3-kinase (C) were measured as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated whether insulin can activate the calcium signaling system in CHO-IR cells. As shown in Fig. 2Go, insulin induced oscillatory elevation of [Ca2+]c in these cells. The effect of insulin was totally dependent on extracellular calcium and blocked by the inorganic calcium channel blockers, nickel or lanthanum. Therefore, insulin elevated [Ca2+]c by stimulating calcium entry. However, 10 nM insulin raised [Ca2+]c in cells, whereas 1 nM insulin induced changes in [Ca2+]c in only a few of them. When cells were first stimulated by 1 nM insulin in medium containing low calcium and the extracellular calcium concentration was raised to 2 mM, elevation of [Ca2+]c was observed in many cells. We interpret these data to mean that 1 nM insulin stimulates calcium entry into most of the cells, but the effect may be too small to be detected by conventional means using fura-2. Because fura-2 is an analog of EGTA, it has considerable capacity to chelate calcium and buffer changes in [Ca2+]c. In fact, when CHO-IR cells were heavily loaded with fura-2, no [Ca2+]c response to insulin was detected (data not shown). The buffering action of fura-2 may minimize the effect of a low dose of insulin on [Ca2+]c. This notion is supported by measuring the transmembrane calcium current, using the patch clamp technique. In any event, a reasonably low concentration of insulin elevated [Ca2+]c in CHO-IR cells; and the effect of insulin was not detected in either parental CHO or CHO-mIR1030 cells. Thus, it is likely that insulin stimulates calcium entry by acting on the insulin receptor. A unique feature of the insulin action in CHO-IR cells is that it is effective in growing (but not in serum-starved) cells. There are at least two explanations for the cell cycle-dependent effect of insulin. First, expression of the insulin-activated channels is reduced in serum-starved cells, so insulin cannot activate them. Second, channels are expressed in serum-starved cells, but the signaling system is attenuated, at some step, in serum-starved cells. When we evaluated the expression of the 10-pS insulin-activated channel in serum-starved cells, by using a cell-attached patch using mastoparan (a direct activator of the G protein) in the pipette, expression of the putative channel was quite low (data not shown). This finding is consistent with the first possibility. Hence, the ability of insulin to stimulate calcium entry greatly depends on the expression of insulin-activated channels in target cells, which may be expressed in a cell cycle-dependent manner. As discussed below, activation of the insulin-activated channel may be necessary for the regulation of cell growth. It is likely that insulin activates the calcium signaling system in growing cells but not in growth-arrested cells.

Patch clamp experiments show that insulin activated voltage-independent calcium-permeable cation channel in CHO-IR cells. This is the first recording of the activation of a single-channel molecule by insulin. The unitary conductance of the insulin-activated channel was 10 pS. The properties of the channel resemble those of the IGF-activated cation channel characterized in Balb/c 3T3 cells (16, 29), but they differ in terms of unitary conductance. In this regard, it is notable that the insulin-activated channel was activated also by IGF-I in CHO-IR cells. When studied in a cell- attached patch, insulin that was added inside the patch activated the channel, whereas that added outside was ineffective. This finding suggests that insulin activates the channel by a direct mechanism, rather than that involving soluble second messengers. Consistent with this notion, the insulin effect was blocked by PTX, and conversely, mastoparan activated the 10-pS channel. Involvement of a G protein in the insulin-induced activation of the channel was directly demonstrated using an excised patch. In these experiments, activation of the channel by the insulin receptor was first confirmed in cell-attached mode. In other words, the receptor, the channel, and the signaling component in the patch were initially confirmed. When the patch was excised and the intracellular surface of the membrane exposed to the bath solution, the channel was inactivated, even in the presence of insulin in the patch pipette, probably because some cytosol components were lost. Insulin-induced activation of the channel was restored by adding GTP, Mg2+, and ATP; and these three are required for the activation of the channel. Because GDP-ßS is inhibitory, these results indicate the involvement of a G protein in the action of insulin. Among G proteins, Ras may not be involved in insulin-induced activation of the channel, because the channel was activated in the presence of anti-Ras antibody. Previous studies have demonstrated that PTX-sensitive G protein is involved in some of the actions of insulin: some of the effects of insulin are blocked by PTX (30, 31, 32, 33); insulin alters ADP ribosylation of the PTX substrate (34); GTP modifies the binding of insulin (35, 36); insulin stimulates association of 41-kDa PTX substrate to the receptor (37); PTX inhibits the activity of insulin receptor kinase (38); and insulin induces unmasking of the C-terminal portion of Gi protein (39). The present results added some insights into the action of insulin by showing that the 10-pS calcium-permeable channel is an effector of the insulin receptor that involves a PTX-sensitive G protein for activation. In addition to GTP and Mg2+, ATP was required to activate the channel by the receptor. Because ATP is not required when the channel is activated by mastoparan, a direct activator of G protein (28), ATP may be necessary for the receptor-mediated activation of the G protein. Interestingly, nonhydrolyzable ATP could substitute for ATP. ATP binding, rather than hydrolysis of ATP, is necessary for the activation of G protein. In our experimental condition, the insulin receptor was first activated by insulin added inside the patch in the cell-attached mode. Presumably, the receptor was autophosphorylated on its tyrosine residues, and conformational changes took place. Then, the patch was excised and exposed to the solution containing GTP, Mg2+, and ATP. It is not totally clear whether the receptor kinase remained in an active state in excised patch. In any event, ATP was still required for the insulin receptor to activate the channel, and nonhydrolyzable ATP could substitute for ATP. This raises the possibility that once activated, the activity of the receptor kinase is not prerequisite, at least for the activation of the G protein. Maddux and Goldfine (40) showed that ATP binding, in the presence of insulin, induces a conformational change in the ß-subunit of the insulin receptor, which may transmit some of the biological signals of insulin. They also showed that AMP-PNP is as effective as ATP (40). They assessed conformational changes in the ß-subunit by measuring the binding of an antibody to the receptor. Their antibody recognized a domain of the ß-subunit located near the major tyrosine autophosphorylation sites at residues 1146, 1150, and 1151. Jo et al. (41) reported that an insulin receptor peptide (1135–1156) stimulates GTP binding to the G protein associated with the insulin receptor. This peptide fulfills the recognition motif for G protein binding (41, 42). Taken together, ATP binding may cause a conformational change in the insulin receptor, so that the putative G protein binding region of the receptor becomes accessible to the G protein. Clearly, further studies are necessary to address this issue. When Tyr-972 of the insulin receptor is mutated to Phe, insulin-mediated phosphorylation of IRS-1 was attenuated (25). Under the same condition, insulin activated the 10-pS calcium-permeable channel. Therefore, phosphorylation of IRS-1 is not a prerequisite for the activation of the channel. In CHO-IR cells, insulin activated the 10-pS calcium-permeable channel. When the activity of this channel was blocked by adding NiCl2, the effect of insulin on DNA synthesis was blocked. The activation of the calcium-permeable channel may be a prerequisite for the insulin-induced stimulation of DNA synthesis. This is consistent with the notion that calcium entry is critical in IGF-I-induced DNA synthesis (16). CHO cells are not a natural target of insulin, and the insulin-induced stimulation of DNA synthesis may not be a physiological action. Nevertheless, the present results indicate, for the first time, that insulin activates the calcium signaling system by opening calcium-permeable channels. They also suggest that insulin-induced activation of the calcium- permeable channel may be important for the growth-promoting action of insulin, at least in some target cells.


   Acknowledgments
 
The authors thank Dr. Kadowaki (of Tokyo University) and Dr. Ebina (of Tokushima University) for providing CHO-IR cells and expression vectors containing mIR972 and mIR1030, respectively. The authors are grateful to Kiyomi Ohgi and Mayumi Odagiri for secretarial assistance during the preparation of the manuscript.


   Footnotes
 
1 The present study was supported by Grant-in-Aid for Scientific Research on Priority Areas from The Ministry of Education, Science, Sports and Culture of Japan. Back

Received June 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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