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Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Smith RM, Baibakov B, Ikebuchi Y, White BH, Lambert NA, Kaczmarek LK, Vogel SS - J. Cell Biol. (2000)

Bottom Line: We tested whether channel distribution can account for the localization of retrieval at exocytotic sites.We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis.Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Georgia, Augusta, Georgia 30912-2630, USA.

ABSTRACT
Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

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P-type channels are inaccessible to toxin before fertilization. Eggs were fertilized in artificial seawater and 100 μM tetramethylrhodamine dextran was added at 5 min after fertilization and net tetramethylrhodamine dextran uptake was determined after a 15-min incubation. All points are normalized to a positive control in the absence of any inhibitor (white bars). Before fertilization some eggs were preincubated with either 500 μM cadmium or 5 μM conotoxin MVIIC for 5 min and then washed to remove free inhibitor (green bars). These eggs were then fertilized and net membrane retrieval was determined. Other eggs in parallel were fertilized in the presence of either cadmium or conotoxin and after three min the eggs were washed to remove free inhibitor (yellow bars). Note the complete inhibition with the irreversible inhibitor, conotoxin. Finally, some eggs were fertilized in the presence of either cadmium or conotoxin with preincubation (red bars) and without preincubation (blue bars), and net membrane retrieval was determined after 15 min. 5 μM conotoxin MVIIC had no effect on sperm-induced fertilization envelope elevation (Vogel et al. 1999). All points are mean ± SD, n = 6 normalized relative to the control.
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Figure 5: P-type channels are inaccessible to toxin before fertilization. Eggs were fertilized in artificial seawater and 100 μM tetramethylrhodamine dextran was added at 5 min after fertilization and net tetramethylrhodamine dextran uptake was determined after a 15-min incubation. All points are normalized to a positive control in the absence of any inhibitor (white bars). Before fertilization some eggs were preincubated with either 500 μM cadmium or 5 μM conotoxin MVIIC for 5 min and then washed to remove free inhibitor (green bars). These eggs were then fertilized and net membrane retrieval was determined. Other eggs in parallel were fertilized in the presence of either cadmium or conotoxin and after three min the eggs were washed to remove free inhibitor (yellow bars). Note the complete inhibition with the irreversible inhibitor, conotoxin. Finally, some eggs were fertilized in the presence of either cadmium or conotoxin with preincubation (red bars) and without preincubation (blue bars), and net membrane retrieval was determined after 15 min. 5 μM conotoxin MVIIC had no effect on sperm-induced fertilization envelope elevation (Vogel et al. 1999). All points are mean ± SD, n = 6 normalized relative to the control.

Mentions: To test if P-type calcium channels might reside in the cortical granule membrane (as opposed to the plasma membrane) before fertilization we took advantage of the fact that ω-conotoxin MVIIC is a functionally irreversible inhibitor of P-type channels (McDonough et al. 1996), whereas cadmium is a readily reversible inhibitor. If the channels are present in the plasma membrane before fertilization, application of ω-conotoxin MVIIC should block them and prevent endocytosis (Vogel et al. 1999). However, if they are inserted into the plasma membrane by exocytosis it should be possible to block them only after the cortical granules have fused. Virtually every cortical granule fuses with the plasma membrane by 3 min after fertilization (Mohri and Hamaguchi 1990; Vogel et al. 1996). We treated eggs before and after fertilization with either cadmium, or with ω-conotoxin MVIIC (Fig. 5). We found that a 5-min pretreatment (green bars) with either cadmium or ω-conotoxin MVIIC followed by washing away unbound inhibitor did not affect membrane retrieval (Fig. 5). In contrast, treatment with ω-conotoxin MVIIC at fertilization and washing 3 min after fertilization (yellow bars) completely blocked endocytotic membrane retrieval. This same protocol with cadmium did not affect membrane retrieval (Fig. 5), as expected if the cadmium block of calcium channels is reversible and membrane retrieval recovers following the transient block. These results also confirm that the ω-conotoxin MVIIC block of calcium channels is functionally irreversible in this system. The fact that a 3-min incubation with ω-conotoxin MVIIC following fertilization completely inhibited membrane retrieval suggests that a 5-min preincubation with toxin before fertilization gave ample time for toxin binding. Even a 15-min preincubation with ω-agatoxin TK, another irreversible P-channel inhibitor, failed to inhibit membrane retrieval (89.1 ± 5.2% retrieval; mean ± SEM, n = 6), while ω-agatoxin TK treatment between 0 and 3 min resulted in an almost complete inhibition of membrane retrieval (9.7 ± 1.9% retrieval; mean ± SEM, n = 6). Thus, if ω-conotoxin MVIIC or ω-agatoxin TK-sensitive channels had been present before fertilization, they should have been blocked by toxin pretreatment. We conclude that the P-type channels required for membrane retrieval are either absent from the egg surface before fertilization or toxin binding is uncoupled from channel gating.


Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Smith RM, Baibakov B, Ikebuchi Y, White BH, Lambert NA, Kaczmarek LK, Vogel SS - J. Cell Biol. (2000)

P-type channels are inaccessible to toxin before fertilization. Eggs were fertilized in artificial seawater and 100 μM tetramethylrhodamine dextran was added at 5 min after fertilization and net tetramethylrhodamine dextran uptake was determined after a 15-min incubation. All points are normalized to a positive control in the absence of any inhibitor (white bars). Before fertilization some eggs were preincubated with either 500 μM cadmium or 5 μM conotoxin MVIIC for 5 min and then washed to remove free inhibitor (green bars). These eggs were then fertilized and net membrane retrieval was determined. Other eggs in parallel were fertilized in the presence of either cadmium or conotoxin and after three min the eggs were washed to remove free inhibitor (yellow bars). Note the complete inhibition with the irreversible inhibitor, conotoxin. Finally, some eggs were fertilized in the presence of either cadmium or conotoxin with preincubation (red bars) and without preincubation (blue bars), and net membrane retrieval was determined after 15 min. 5 μM conotoxin MVIIC had no effect on sperm-induced fertilization envelope elevation (Vogel et al. 1999). All points are mean ± SD, n = 6 normalized relative to the control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169375&req=5

Figure 5: P-type channels are inaccessible to toxin before fertilization. Eggs were fertilized in artificial seawater and 100 μM tetramethylrhodamine dextran was added at 5 min after fertilization and net tetramethylrhodamine dextran uptake was determined after a 15-min incubation. All points are normalized to a positive control in the absence of any inhibitor (white bars). Before fertilization some eggs were preincubated with either 500 μM cadmium or 5 μM conotoxin MVIIC for 5 min and then washed to remove free inhibitor (green bars). These eggs were then fertilized and net membrane retrieval was determined. Other eggs in parallel were fertilized in the presence of either cadmium or conotoxin and after three min the eggs were washed to remove free inhibitor (yellow bars). Note the complete inhibition with the irreversible inhibitor, conotoxin. Finally, some eggs were fertilized in the presence of either cadmium or conotoxin with preincubation (red bars) and without preincubation (blue bars), and net membrane retrieval was determined after 15 min. 5 μM conotoxin MVIIC had no effect on sperm-induced fertilization envelope elevation (Vogel et al. 1999). All points are mean ± SD, n = 6 normalized relative to the control.
Mentions: To test if P-type calcium channels might reside in the cortical granule membrane (as opposed to the plasma membrane) before fertilization we took advantage of the fact that ω-conotoxin MVIIC is a functionally irreversible inhibitor of P-type channels (McDonough et al. 1996), whereas cadmium is a readily reversible inhibitor. If the channels are present in the plasma membrane before fertilization, application of ω-conotoxin MVIIC should block them and prevent endocytosis (Vogel et al. 1999). However, if they are inserted into the plasma membrane by exocytosis it should be possible to block them only after the cortical granules have fused. Virtually every cortical granule fuses with the plasma membrane by 3 min after fertilization (Mohri and Hamaguchi 1990; Vogel et al. 1996). We treated eggs before and after fertilization with either cadmium, or with ω-conotoxin MVIIC (Fig. 5). We found that a 5-min pretreatment (green bars) with either cadmium or ω-conotoxin MVIIC followed by washing away unbound inhibitor did not affect membrane retrieval (Fig. 5). In contrast, treatment with ω-conotoxin MVIIC at fertilization and washing 3 min after fertilization (yellow bars) completely blocked endocytotic membrane retrieval. This same protocol with cadmium did not affect membrane retrieval (Fig. 5), as expected if the cadmium block of calcium channels is reversible and membrane retrieval recovers following the transient block. These results also confirm that the ω-conotoxin MVIIC block of calcium channels is functionally irreversible in this system. The fact that a 3-min incubation with ω-conotoxin MVIIC following fertilization completely inhibited membrane retrieval suggests that a 5-min preincubation with toxin before fertilization gave ample time for toxin binding. Even a 15-min preincubation with ω-agatoxin TK, another irreversible P-channel inhibitor, failed to inhibit membrane retrieval (89.1 ± 5.2% retrieval; mean ± SEM, n = 6), while ω-agatoxin TK treatment between 0 and 3 min resulted in an almost complete inhibition of membrane retrieval (9.7 ± 1.9% retrieval; mean ± SEM, n = 6). Thus, if ω-conotoxin MVIIC or ω-agatoxin TK-sensitive channels had been present before fertilization, they should have been blocked by toxin pretreatment. We conclude that the P-type channels required for membrane retrieval are either absent from the egg surface before fertilization or toxin binding is uncoupled from channel gating.

Bottom Line: We tested whether channel distribution can account for the localization of retrieval at exocytotic sites.We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis.Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Georgia, Augusta, Georgia 30912-2630, USA.

ABSTRACT
Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Show MeSH
Related in: MedlinePlus