TRANSLATIONAL PHYSIOLOGY
Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

Lukas Schwake1,*, Andreas W. Henkel2,*, Hans D. Riedel1, Thorsten Schlenker1, Matthias Both2, Andrea Migala2, Boris Hadaschik1, Nataly Henfling1, and Wolfgang Stremmel1

1 Department of Gastroenterology, Faculty of Medicine, University of Heidelberg, D-69115 Heidelberg; and 2 Department of Biomedical Optics, Max-Planck-Institute for Medical Research, D-69120 Heidelberg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.

hereditary hemochromatosis; transferrin receptor; patch-clamp capacitance measurements


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RECEPTOR-MEDIATED ENDOCYTOSIS of transferrin-bound iron [diferric transferrin (Tf)] is supposed to be the principal mechanism of iron uptake by nonintestinal cells (29). Binding of Tf to the transferrin receptor (TfR) induces rapid formation of clathrin-coated pits and vesicles. After endocytosis of the Tf-TfR complex, iron is released from Tf by a decrease in endosomal pH and enters the chelatable intracellular iron pool (11, 25, 35). Recent investigations (9, 13, 26, 27, 31) indicate a key role of HFE, the hereditary hemochromatosis gene product, in regulating endocytosis of Tf. Hereditary hemochromatosis, one of the most common genetic disorders overall, is characterized by an upregulation of intestinal iron absorption and increased iron deposition in the cytoplasm of parenchymal cells, causing tissue damage and organ failure (14, 29). HFE, the responsible gene, was identified in 1996 (8). Its gene product is expressed in many organs, with the highest levels detected in liver and small intestine (9, 20). Most patients with hereditary hemochromatosis are homozygous for the C282Y mutation, which is characterized by the substitution of tyrosine for cysteine at amino acid 282. This alteration causes the loss of a disulfide bond in the alpha 3 domain of HFE, resulting in improper folding, loss of cell surface expression, and lack of association with beta 2-microglobulin (8, 10, 37). Because wild-type HFE colocalizes with the TfR at the cell surface and in various intracellular compartments (9, 16, 26, 30, 33), a close functional interaction between both proteins is predicted. The knockout of the HFE gene has been shown to produce a valid mouse model of hereditary hemochromatosis (38). Furthermore, HeLa cells overexpressing wild-type HFE have shown decreased intracellular ferritin levels (16, 27), reduced incorporation of 55Fe-transferrin (7, 31), and slightly increased TfR levels (16, 27). Calculation of the Tf-TfR internalization rate in transfected HeLa cells has suggested inhibition of Tf internalization by HFE binding to the TfR (33). To further study the functional interaction of HFE and TfR during the time course of endocytosis, we applied patch-clamp capacitance measurements to transfected HeLa cells expressing wild-type or C282Y-mutant HFE under the control of a tetracycline-sensitive promoter (15, 27). Endo- and exocytosis during transient exposure to Tf were monitored in single cells by whole cell and cell-attached capacitance techniques using sinusoidal excitation and a lock-in amplifier (1, 3, 23, 24).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and solutions. The stably transfected HeLa cell line HtTA-HFE expressing HFE under the control of a tetracycline-sensitive promoter ("tet-off" system) was generated as previously described (15). Briefly, plasmid pSGH-1, which contains the HFE encoding region under the control of a tetracycline-sensitive promoter, was stably transfected into the HeLa cell line HtTA, which constitutively expresses the tetracycline-dependent transactivator tTA (27). Subconfluent HtTA-HFE cells grown in the presence [HFE(-)] or absence [HFE(+)] of 1 µg/ml doxycycline were used for patch-clamp studies 2-4 days after cells were plated. HtTA-HFE282 cells carrying the C282Y-mutant HFE cDNA were generated by transfecting HtTA with a mutated derivative of pSGH-1. Western blot analyses of HtTA cells transfected with wild-type or C282Y-mutant HFE revealed that both proteins are expressed in comparable amounts. Immunohistochemistry demonstrated a similar intracellular distribution of both proteins, whereas only wild-type HFE was detectable at the cell surface (Riedel HD, unpublished observations). In the presence of doxycycline, the amounts of TfR were comparable in HtTA-HFE wild-type and HtTA-HFE282 cells, whereas in the absence of doxycycline, TfR was about twofold increased in HFE-HtTA wild-type cells, as previously shown (27). This increase was not observed in HFE-HtTA282 cells (Riedel HD, unpublished observations). The standard bath medium (extracellular) was composed of (in mM) 150 NaCl, 2 KCl, 2 CaCl2, 2 MgCl2, and 20 HEPES (pH 7.2, 320 mosmol). The pipette solution (intracellular) contained (in mM) 100 CsGlu, 20 NaCl, 3 MgCl2, 0.1 CaCl2, 0.2 EGTA, and 40 HEPES (pH 7.2, 297 mosmol). Experiments were performed at room temperature. Diferric (holo-) transferrin (Sigma, St. Louis, MO), nonferric (apo-) transferrin (Sigma), and human albumin (Sigma) were used at a concentration of 10 µg/ml.

Pipettes and patch-clamp sealing. Patch pipettes (borosilicate glass, wall thickness 0.38 mm, outer diameter 1.5 mm) from WPI (Sarasota, FL) were pulled on a model P-97 Flaming/Brown micropipette puller (Sutter Instrument, Novacota, CA), coated with type 2356200 dental wax (Schmalz, Heidelberg, Germany), and fire-polished to a final resistance of 1.5-3 MOmega . During whole cell and cell-attached measurements, the membrane potential was clamped to -40 mV.

Capacitance measurements. In whole cell configuration, the plasma membrane capacitance was measured with an SR830-DSP two-phase lock-in amplifier (Stanford Research Systems, Stanford, CA) by superimposing an 800-Hz sinusoidal voltage (60 mV peak to peak) onto the holding potential of -40 mV (36). An EPC-7 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany) was connected to the lock-in amplifier. The lock-in amplifier analyzed the resulting current that was phase-shifted with respect to the command voltage. This relation can be expressed as the cell's complex admittance, which has a real part (Re) corresponding to the conductance and an imaginary, 90° out-of-phase part (Im) corresponding to the capacitance of the cell. For whole cell measurements, cell capacitance was recorded for 5-15 min at a sampling frequency of 50 Hz. Data were collected via an analog-to-digital/digital-to-analog converter (Labmaster TL-1 DMA; Axon Instruments, Foster City, CA) into a 166-MHz Pentium computer running Windows 95. The recording (LabMaster Recorder 2.05) and analyzing (WinPCA 3.5) software was written by one of the authors (A. W. Henkel). In cell-attached configuration, an 8-kHz sine wave of 280 mV peak to peak was applied to the patch membrane, and the data points were sampled at 350 Hz. Phase settings for cell-attached measurements were done as recently described (17). In whole cell recordings, we adjusted the phase angle by short, repetitive resistance changes between the bath electrode and the EPC-7 head stage until no deflections were seen in the capacitance trace. To optimize the phase angle after recording, we employed the capacitance noise-minimization technique analogous to cell-attached recordings.

Data analysis. Fast capacitance steps in cell-attached measurements were detected by using an automated algorithm, as previously described (17). Only solitary steps with amplitudes between 0.15 and 2 femtofarads (fF) were included in our data set when the capacitance noise within 150-ms sections before and after the step was <50 attofarads (aF). Steps recorded during periods with higher capacitance noise were not included in the analyses. Slow capacitance changes in whole cell recordings that occurred during perfusion of the cell with diferric transferrin or standard bath medium, respectively, were analyzed by dividing the capacitance trace into sections of 5 s. The net capacitance change (Delta C), which equals the speed of endocytosis or exocytosis, was measured in each section, and the resulting data were plotted in a histogram. Fast capacitance changes that occurred sometimes after switching between perfusion solutions were attributed to artificial pressure effects. Such sections did not influence the peak position of the distribution histogram.

Statistics. Significance of differences between mean values of two groups in whole cell and cell-attached experiments was tested by using Student's paired or unpaired t-test. Significance was attributed at P < 0.01. Peak parameter determination in histograms was made with nonlinear curve fitting using Microcal Origin's (version 4.1) built-in Gaussian peak function.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transferrin-induced capacitance changes revealed by whole cell measurements. Whole cell experiments were performed by using HeLa cells with suppressed expression of wild-type HFE [HFE(-)], HeLa cells overexpressing wild-type HFE [HFE(+)], and HeLa cells overexpressing C282Y-mutant HFE [C282Y(+)]. Capacitance changes detected in whole cell experiments reflect the net effect of all endo- and exocytic events in one cell, whereas cell-attached measurements are restricted to the plasma membrane encompassed by the pipette (Fig. 1). To evaluate the effect of Tf on the endocytic activity of single HeLa cells, we perfused cells with standard bath medium (SBM) with or without added Tf, and whole cell capacitance was measured simultaneously. Irregularly shaped cells were excluded from analysis to reduce the impact of cellular morphology on the reliability of capacitance measurements.


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Fig. 1.   Patch-clamp capacitance techniques. A: whole cell configuration. Cells were perfused alternately with pure standard bath medium (SBM) or with SBM with added diferric transferrin (Tf) using a microperfusion system. In selected experiments SBM was supplemented with human albumin (HA) or apo-transferrin instead of Tf in equimolar concentration. B: cell-attached configuration. The patch-pipette was filled with pure SBM, SBM with added Tf, or SBM with HA. All experiments were performed at room temperature.

Figure 2 depicts a representative whole cell recording from three single HeLa cells using Tf as a "stimulator" of endocytosis. The downward drift of the capacitance trace represents a decrement in membrane capacitance and results from a relative predominance of endocytosis caused by either increased endocytic or decreased exocytic activity. In contrast, the upward drift of the capacitance trace reflects an increment in membrane capacitance due to a relative predominance of exocytosis. As shown in Fig. 2A with a single HFE(-) cell, we consistently observed a decrement in whole cell membrane capacitance in the presence of Tf. This effect was not detected when the cell was perfused with pure SBM. Interestingly, no predominance of endocytosis was observed in the HFE(+) cell during Tf perfusion (Fig. 2B). Figure 2C shows the effect of Tf on a representative HeLa cell overexpressing C282Y-mutant HFE. Tf perfusion still caused a decrease in membrane capacitance; however, changes were less pronounced compared with the HFE(-) cell. To further quantify changes in membrane capacitance from HFE(-), HFE(+), and C282Y(+) cells, capacitance traces were divided into sections of 5 s and Delta C values were then determined. Positive capacitance changes indicate periods with a relative predominance of exocytic activity, whereas negative capacitance changes reflect predominating endocytosis. Figure 3 shows the results from a typical HFE(-) cell. As shown by mainly negative bins, perfusion with Tf resulted in a predominance of endocytosis (Fig. 3A). In contrast, after perfusion with Tf-free SBM, we observed mainly periods with a predominance of exocytosis in this cell (Fig. 3B).


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Fig. 2.   Capacitance trace (Delta C) detected by whole cell measurements. Characteristic capacitance traces from transfected HeLa cells were detected by whole cell measurements. Results shown are from a single HeLa cell with suppressed expression of wild-type HFE [HFE(-)] (A), a single HeLa cell expressing wild-type HFE [HFE(+)] (B), and a single HeLa cell expressing C282Y-mutant HFE [C282Y(+)] (C). Each cell was exposed several times to SBM containing either no Tf or Tf at a concentration of 10 µg/ml. HFE, hereditary hemochromatosis protein.



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Fig. 3.   Quantification of Tf-induced capacitance changes. Histograms show plots of capacitance changes from a single HFE(-) cell as determined by whole cell measurements. Sections of 5 s were analyzed and summarized during perfusion with SBM and added Tf at a concentration of 10 µg/ml (A) or during perfusion with pure SBM (B). Negative values indicate predominant endocytic capacitance changes, whereas positive values indicate predominant exocytic capacitance changes in the respective sections. Error bars indicate the square root of the number of sections in each bin.

Predominance of endocytosis during Tf perfusion in HFE(-) and C282Y(+) cells but not in HFE(+) cells. Figure 4 summarizes the results of whole cell capacitance measurements in HFE(-), HFE(+), and C282Y(+) cells. We observed a clear net decrease in membrane capacitance when HFE(-) cells were exposed to Tf. In contrast, no significant capacitance decrease was detected in HFE(+) cells during perfusion with Tf. As shown in Fig. 4, the net decrease in membrane capacitance in C282Y(+) cells during perfusion with Tf was significantly different from measurements in both HFE(-) and HFE(+) cells. Compared with HFE(-) cells, C282Y(+) cells were characterized by a reduced decrease in membrane capacitance, reflecting only a moderate predominance of endocytosis during transient exposure to Tf. To exclude nonspecific effects of Tf on endocytosis, we performed several control experiments. In selected studies in which C282Y(+) cells were used, Tf was substituted with apotransferrin (apo-Tf) or human albumin (HA). Perfusion of single C282Y(+) cells with these proteins, however, did not significantly alter their membrane capacitance (data not shown).


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Fig. 4.   Tf-induced endocytosis in HFE(-), HFE(+), and C282Y(+) cells. Graph summarizes mean endocytic and exocytic activity measured by whole cell capacitance technique during perfusion with pure SBM or SBM with added Tf. Negative values indicate mean endocytic rates, whereas positive values indicate mean exocytic rates in 38 HFE(-) cells, 27 HFE(+) cells, and 27 C282Y(+) cells. Error bars indicate SE of the mean. Rates in HFE(-) and C282Y(+) cells differ significantly from rates in HFE(+) cells during perfusion with Tf (P < 0.01). Moreover, mean endocytic rates are also significantly different between C282Y(+) and HFE(-) cells (P < 0.01).

Cell-attached recordings detect formation of single endo- and exocytic vesicles. To more specifically characterize the effect of Tf on endocytosis, we performed cell-attached capacitance measurements in HFE(-) and HFE(+) cells. Capacitance and conductance of the membrane patch encompassed by the pipette were recorded simultaneously to detect single endo- and exocytic vesicles. Figure 5 depicts a typical experiment with a single HFE(-) cell in which Tf is present inside the pipette. Downward steps in the capacitance trace indicate the separation of single endocytic vesicles from the membrane patch under the pipette. In contrast, upward steps correspond to the formation of single exocytic vesicles. The latter were not observed in this trace. As illustrated in Fig. 5, downward steps in the capacitance trace had different amplitudes, although most of them were <0.3 fF.


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Fig. 5.   Endocytic vesicles detected by cell-attached recordings. Representative capacitance trace (Delta C) was recorded from a single HFE(-) cell in cell-attached configuration exposed to Tf (inside the pipette). Several downward steps in capacitance correspond to the formation of endocytic vesicles. The Delta G trace corresponds to the conductance of the cell.

Transferrin stimulates formation of endocytic vesicles in HFE(-) cells. Cell-attached recordings were made from a total of 126 HFE(+) and 164 HFE(-) cells. Total recording time was 15.3 h for HFE(-) cells and 11.0 h for HFE(+) cells. The frequency of endo- and exocytic events was calculated as the number of endo- and exocytic steps per 1,000 s and was specified in milli-Hertz (mHz) for each cell. The patch-clamp pipette was filled with pure SBM or SBM containing Tf or HA in equimolar concentration. Figure 6 summarizes the frequencies of endocytic events in the cell lines under the specified conditions. HA did not increase the basal rate of endocytosis in either HFE(+) or HFE(-) cells. However, in HFE(-) cells we observed an approximately fourfold increase in endocytic vesicle formation when the patch was exposed to Tf. This effect was not detected in HFE(+) cells. The number of exocytic vesicles detected was independent of pipette contents in both HeLa cell lines (data not shown).


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Fig. 6.   Step frequency of endocytic vesicles in HFE(-) and HFE(+) cells. Bar graph shows the summary of endocytic step counts observed during cell-attached recordings from HFE(-) and HFE(+)cells. The step frequency is indicated as endocytic vesicles per 1,000 s of recording time (milli-Hertz). Experiments were performed with pure SBM, SBM with added Tf, or SBM with added HA. The numbers of cells analyzed were 78 with HA, 54 with Tf, and 32 with pure SBM for HFE(-) cells and 27 with HA, 50 with Tf, and 49 with pure SBM for HFE(+)cells. The Tf-induced endocytic step frequency in HFE(-) cells was significantly increased compared with all other experimental conditions (* P < 0.01).

Cell-attached data allowed the size of endocytic vesicles to be calculated. As shown in previous studies, the amplitude of the capacitance steps shown in Fig. 5 can be used to calculate the vesicle size (17). We used a specific capacitance of 10 fF/µm2 to quantify vesicle size distribution (5). Figure 7 shows the step-size histogram for all analyzed HFE(-) and HFE(+) cells exposed to Tf. Both cell clones were characterized by a predominance of endocytic vesicles; however, this effect was more prominent in HFE(-) cells. Whereas the total number of endocytic vesicles differed largely in HFE(-) and HFE(+) cells, the distribution of vesicle diameter was in the same range. Many of the identified endocytic vesicles had calculated diameters between 100 and 150 nm; however, some smaller and larger vesicles were also observed.


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Fig. 7.   Distribution of endocytic and exocytic vesicles according to their diameters. Vesicle size histogram shows results from cell-attached experiments with Tf (inside the pipette). Negative diameters are plotted for endocytic vesicles and positive diameters for exocytic vesicles. Specific capacitance used was 10 fF/µm2. Vesicles are sorted in 10-nm bins. Results from HFE(-) cells (n = 54) (A) are compared with results from HFE(+) cells (n = 50) (B). Error bars indicate the square root of the step count. Many vesicles are ~100 nm in diameter and may refer to clathrin-coated vesicles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cellular iron uptake by the TfR pathway and its regulation by HFE, the hereditary hemochromatosis gene product, is a complex and only partially understood process (11, 29). Previous work has suggested that the expression of wild-type HFE impairs Tf uptake and reduces the affinity of TfR for its ligand Tf (9, 16, 27, 31, 32). However, because of methodological limitations of kinetic analyses, no direct time-resolved measurements of the endocytic uptake of Tf and the effect of HFE on this process have been performed to date. To resolve this problem, we determined Tf-induced endocytosis by patch-clamp capacitance techniques in stably transfected HeLa cells expressing recombinant wild-type or mutant HFE under the control of a tetracycline sensitive promoter (HtTA-HFE and HtTA-HFE282). In these cells, HFE synthesis can be tightly controlled (27). Specifically, addition of doxycycline almost completely inhibits synthesis of recombinant HFE and allows regulation of HFE synthesis by more than two orders of magnitude. In addition, in the HeLa cell line HtTA, no expression of HFE can be detected by Western blotting (27), and even with highly sensitive RT-PCR, only a faint HFE-specific product is detectable (Riedel HD, unpublished observations). Stably transfected HeLa cells expressing HFE under the control of a tetracycline-sensitive promoter therefore provide a suitable cellular model system for studying HFE function and have been used in a number of recently published studies (7, 9, 16, 27, 30, 32).

Whole cell and cell-attached capacitance techniques enabled us to measure the extent and rate of endocytosis as well as the size and kinetics of single endocytic vesicles. In the past, these electrophysiological methods were mainly applied to excitable neuronal cells (2, 17, 19), neutrophils (22, 23), eosinophils (34), and mast cells (4, 12). To our knowledge, these techniques have not previously been used to study Tf-induced endocytosis.

Results from whole cell experiments in HeLa cells with suppressed expression of wild-type HFE and in HeLa cells overexpressing C282Y-mutant HFE revealed a decrease in membrane capacitance in the presence of Tf. In fact, this effect, which reflects a predominance of endocytosis, was more pronounced in HeLa cells with suppressed expression of wild-type HFE than in HeLa cells overexpressing C282Y-mutant HFE. In contrast, membrane capacitance remained essentially unaffected when Tf was applied to HeLa cells overexpressing wild-type HFE. These findings indicate that wild-type HFE acts as a negative modulator of Tf-induced endocytosis. They are consistent with results from kinetic analyses, which showed a decreased uptake of labeled Tf in cells overexpressing wild-type HFE (33).

The observation that C282Y-mutant HeLa cells still show a moderate predominance of endocytosis indicates that this mutation does not completely abolish HFE function. Although it has been reported that C282Y-mutant HFE cannot be exposed at the cell surface in association with TfR (10, 37), it still preserves its ability to modulate endocytosis in the presence of Tf. Results from transgenic and knockout mice support this hypothesis. Consistent with our findings, iron overload in mice carrying the artificially induced C282Y mutation appears to be less severe than in HFE knockout mice (21).

A net capacitance decrease observed in whole cell experiments represents a relative predominance of endocytosis and may result from increased endocytic activity as well as from decreased exocytic activity. To obtain more information about this process, we performed further experiments in cell-attached configuration using HeLa cells overexpressing wild-type HFE and HeLa cells with suppressed expression of wild-type HFE. Single endo- and exocytic steps can be detected using this method, which is ~100 times more sensitive than the whole cell configuration. Increased formation of endocytic vesicles in the presence of Tf was observed only in HeLa cells with suppressed expression of wild-type HFE, not in HeLa cells overexpressing wild-type HFE. These findings clearly indicate that the net decrease in membrane capacitance observed in whole cell recordings indeed reflects increased endocytic activity rather than decreased exocytic activity. Nonspecific protein effects on endocytosis are highly unlikely, because increased formation of endocytic vesicles was not observed when HA was used in the pipette rather than Tf.

Results from cell-attached experiments allowed us to calculate the size of endo- and exocytic vesicles. Many of the identified endocytic vesicles had calculated diameters between 100 and 150 nm (Fig. 7) and thus may correspond to clathrin-coated vesicles (28). These vesicles are the principal organelles for the endocytic uptake of Tf-TfR complexes (6, 11). However, smaller and larger endocytic vesicles were also observed. Furthermore, because of the limited resolution of cell-attached measurements, vesicles of <80 nm could not be detected by our automated algorithm.

Recent findings suggesting that wild-type HFE accumulates nonfunctional TfR at the cell surface (33) support the hypothesis that HFE directly affects the classic pathway of TfR-mediated endocytosis of Tf-bound iron. A decrease in TfR affinity, as shown by others (9, 16, 18), may contribute to this effect.

Taken together, our results from patch-clamp capacitance measurements indicate that HFE effectively suppresses the endocytic uptake of Tf-TfR complexes. This effect is attenuated, but not completely abolished, in HeLa cells expressing C282Y-mutant HFE. Moreover, we can show that Tf specifically stimulates endocytosis in stably transfected HeLa cells. Finally, as demonstrated in the present study, patch-clamp capacitance techniques provide a suitable methodological approach to study Tf-induced endocytosis and its regulation by HFE, the hereditary hemochromatosis gene product.


    ACKNOWLEDGEMENTS

We thank Brenda Stride for critical reading of the manuscript.


    FOOTNOTES

* L. Schwake and A. W. Henkel contributed equally to this work.

This work was supported by the Deutsche Forschungsgemeinschaft (STR216) and the Dietmar Hopp Foundation.

Address for reprint requests and other correspondence: L. Schwake, Dept. of Internal Medicine (Gastroenterology), Faculty of Medicine, Univ. of Heidelberg, Bergheimer-Strasse 58, D-69115 Heidelberg, Germany (E-mail: Lukas_Schwake{at}gmx.net).

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

First published January 2, 2002;10.1152/ajpcell.00415.2001

Received 27 August 2001; accepted in final form 13 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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