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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 3
domain of HFE, resulting in improper folding, loss of cell surface
expression, and lack of association with
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M. 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 (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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
|
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).
|
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.
|
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).
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Albillos, A,
Dernick G,
Horstmann H,
Almers W,
Alvarez de Toledo G,
and
Lindau M.
The exocytotic event in chromaffin cells revealed by patch amperometry.
Nature
389:
509-512,
1997[ISI][Medline].
2.
Alés, E,
Tabares L,
Poyato JM,
Valero V,
Lindau M,
and
Alvarez de Toledo G.
High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.
Nat Cell Biol
1:
40-44,
1999[ISI][Medline].
3.
Almers, W,
and
Neher E.
Gradual and stepwise changes in the membrane capacitance of rat peritoneal mast cells.
J Physiol
386:
205-217,
1987[Abstract].
4.
Breckenridge, LJ,
and
Almers W.
Currents through the fusion pore that forms during exocytosis of a secretory vesicle.
Nature
328:
814-817,
1987[ISI][Medline].
5.
Cole, KS.
Membranes, ions and impulses. Berkeley, CA: Univ. of California Press, 1968, p. 1.
6.
Conrad, ME,
Umbreit JN,
and
Moore EG.
Iron absorption and transport.
Am J Med Sci
318:
213-229,
1999[ISI][Medline].
7.
Corsi, B,
Levi S,
Cozzi A,
Corti A,
Altimare D,
Albertini A,
and
Arosio P.
Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces an iron-deficient phenotype.
FEBS Lett
460:
149-152,
1999[ISI][Medline].
8.
Feder, JN,
Gnirke A,
Thomas W,
Tsuchihashi Z,
Ruddy DA,
Basava A,
Dormishian F,
Domingo R, Jr,
Ellis MC,
Fullan A,
Hinton LM,
Jones NL,
Kimmel BE,
Kronmal GS,
Lauer P,
Lee VK,
Loeb DB,
Mapa FA,
McClelland E,
Meyer NC,
Mintier GA,
Moeller N,
Moore T,
Morikang E,
Wolff RK,
A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.
Nat Genet
13:
399-408,
1996[ISI][Medline].
9.
Feder, JN,
Penny DM,
Irrinki A,
Lee VK,
Lebron JA,
Watson N,
Tsuchihashi Z,
Sigal E,
Bjorkman PJ,
and
Schatzman RC.
The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.
Proc Natl Acad Sci USA
95:
1472-1477,
1998
10.
Feder, JN,
Tsuchihashi Z,
Irrinki A,
Lee VK,
Mapa FA,
Morikang E,
Prass CE,
Starnes SM,
Wolff RK,
Parkkila S,
Sly WS,
and
Schatzman RC.
The hemochromatosis founder mutation in HLA-H disrupts 2-microglobulin interaction and cell surface expression.
J Biol Chem
272:
14025-14028,
1997
11.
Feelders, RA,
Kuiper-Kramer EP,
and
van Eijk HG.
Structure, function and clinical significance of transferrin receptors.
Clin Chem Lab Med
37:
1-10,
1999[ISI][Medline].
12.
Fernandez, JM,
Neher E,
and
Gomperts BD.
Capacitance measurements reveal stepwise fusion events in degranulating mast cells.
Nature
312:
453-455,
1984[ISI][Medline].
13.
Fleming, RE,
Migas MC,
Holden CC,
Waheed A,
Britton RS,
Tomatsu S,
Bacon BR,
and
Sly WS.
Transferrin receptor 2: continued expression in mouse liver in the face of iron overload and in hereditary hemochromatosis.
Proc Natl Acad Sci USA
97:
2214-2219,
2000
14.
Fleming, RE,
Migas MC,
Zhou X,
Jiang J,
Britton RS,
Brunt EM,
Tomatsu S,
Waheed A,
Bacon BR,
and
Sly WS.
Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increased duodenal expression of the iron transporter DMT1.
Proc Natl Acad Sci USA
96:
3143-3148,
1999
15.
Gossen, M,
and
Bujard H.
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
Proc Natl Acad Sci USA
89:
5547-5551,
1992[Abstract].
16.
Gross, CN,
Irrinki A,
Feder JN,
and
Enns CA.
Co-trafficking of HFE, a nonclassical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation.
J Biol Chem
273:
22068-22074,
1998
17.
Henkel, AW,
Meiri H,
Horstmann H,
Lindau M,
and
Almers W.
Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells.
EMBO J
19:
84-93,
2000
18.
Ikuta, K,
Fujimoto Y,
Suzuki Y,
Tanaka K,
Saito H,
Ohhira M,
Sasaki K,
and
Kohgo Y.
Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells.
Biochim Biophys Acta
1496:
221-231,
2000[ISI][Medline].
19.
Kibble, AV,
Barnard RJ,
and
Burgoyne RD.
Patch-clamp capacitance analysis of the effects of -SNAP on exocytosis in adrenal chromaffin cells.
J Cell Sci
109:
2417-2422,
1996
20.
Lebron, JA,
West AP, Jr,
and
Bjorkman PJ.
The hemochromatosis protein HFE competes with transferrin for binding to the transferrin receptor.
J Mol Biol
294:
239-245,
1999[ISI][Medline].
21.
Levy, JE,
Montross LK,
Cohen DE,
Fleming MD,
and
Andrews NC.
The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.
Blood
94:
9-11,
1999
22.
Lollike, K,
Borregaard N,
and
Lindau M.
The exocytotic fusion pore of small granules has a conductance similar to an ion channel.
J Cell Biol
129:
99-104,
1995[Abstract].
23.
Lollike, K,
and
Lindau M.
Membrane capacitance techniques to monitor granule exocytosis in neutrophils.
J Immunol Methods
232:
111-120,
1999[ISI][Medline].
24.
Neher, E,
and
Marty A.
Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.
Proc Natl Acad Sci USA
79:
6712-6716,
1982[Abstract].
25.
Ponka, P,
and
Lok CN.
The transferrin receptor: role in health and disease.
Int J Biochem Cell Biol
31:
1111-1137,
1999[ISI][Medline].
26.
Ramalingam, TS,
West AP, Jr,
Lebron JA,
Nangiana JS,
Hogan TH,
Enns CA,
and
Bjorkman PJ.
Binding to the transferrin receptor is required for endocytosis of HFE and regulation of iron homeostasis.
Nat Cell Biol
2:
953-957,
2000[ISI][Medline].
27.
Riedel, HD,
Muckenthaler MU,
Gehrke SG,
Mohr I,
Brennan K,
Herrmann T,
Fitscher BA,
Hentze MW,
and
Stremmel W.
HFE downregulates iron uptake from transferrin and induces iron-regulatory protein activity in stably transfected cells.
Blood
94:
3915-3921,
1999
28.
Robinson, MS,
Watts C,
and
Zerial M.
Membrane dynamics in endocytosis.
Cell
84:
13-21,
1996[ISI][Medline].
29.
Rolfs, A,
and
Hediger MA.
Metal ion transporters in mammals: structure, function and pathological implications.
J Physiol
518:
1-12,
1999
30.
Roy, CN,
Carlson EJ,
Anderson EL,
Basava A,
Starnes SM,
Feder JN,
and
Enns CA.
Interactions of the ectodomain of HFE with the transferrin receptor are critical for iron homeostasis in cells.
FEBS Lett
484:
271-274,
2000[ISI][Medline].
31.
Roy, CN,
Penny DM,
Feder JN,
and
Enns CA.
The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells.
J Biol Chem
274:
9022-9028,
1999
32.
Salter-Cid, L,
Brunmark A,
Li Y,
Leturcq D,
Peterson PA,
Jackson MR,
and
Yang Y.
Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis.
Proc Natl Acad Sci USA
96:
5434-5439,
1999
33.
Salter-Cid, L,
Brunmark A,
Peterson PA,
and
Yang Y.
The major histocompatibility complex-encoded class I-like HFE abrogates endocytosis of transferrin receptor by inducing receptor phosphorylation.
Genes Immun
1:
409-417,
2000[ISI][Medline].
34.
Scepek, S,
Coorssen JR,
and
Lindau M.
Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms.
EMBO J
17:
4340-4345,
1998
35.
Seaman, MN,
Burd CG,
and
Emr SD.
Receptor signalling and the regulation of endocytic membrane transport.
Curr Opin Cell Biol
8:
549-556,
1996[ISI][Medline].
36.
Thomas, P,
Surprenant A,
and
Almers W.
Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary.
Neuron
5:
723-733,
1990[ISI][Medline].
37.
Waheed, A,
Parkkila S,
Zhou XY,
Tomatsu S,
Tsuchihashi Z,
Feder JN,
Schatzman RC,
Britton RS,
Bacon BR,
and
Sly WS.
Hereditary hemochromatosis: effects of C282Y and H63D mutations on association with 2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells.
Proc Natl Acad Sci USA
94:
12384-12389,
1997
38.
Zhou, XY,
Tomatsu S,
Fleming RE,
Parkkila S,
Waheed A,
Jiang J,
Fei Y,
Brunt EM,
Ruddy DA,
Prass CE,
Schatzman RC,
O'Neill R,
Britton RS,
Bacon BR,
and
Sly WS.
HFE gene knockout produces mouse model of hereditary hemochromatosis.
Proc Natl Acad Sci USA
95:
2492-2497,
1998