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Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

Liu Y, Schirra C, Edelmann L, Matti U, Rhee J, Hof D, Bruns D, Brose N, Rieger H, Stevens DR, Rettig J - J. Cell Biol. (2010)

Bottom Line: Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin.Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps.Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Physiologie, Universität des Saarlandes, 66421 Homburg, Germany.

ABSTRACT
Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

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The releasable pools in CAPS DKO cells expressing open syntaxin are rapidly exhausted. (A) The free calcium concentration (top) and the capacitance change (bottom) are shown. Those cells expressing open syntaxin exhibit very strong secretion (n = 29) compared with CAPS DKO cells not expressing open syntaxin (n = 27). (B) To determine the amount of secretion remaining after the calcium ramp stimulation, a flash was applied 3 s after the ramp ended to increase calcium to high levels. The residual secretion in the CAPS DKO cells was larger than that of the DKO cells expressing open syntaxin and accounted for ∼65% of the total secretion, whereas the flash response in the open syntaxin–expressing CAPS DKO cells accounted for ∼25% of the total secretion. Error bars indicate mean ± SEM.
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fig3: The releasable pools in CAPS DKO cells expressing open syntaxin are rapidly exhausted. (A) The free calcium concentration (top) and the capacitance change (bottom) are shown. Those cells expressing open syntaxin exhibit very strong secretion (n = 29) compared with CAPS DKO cells not expressing open syntaxin (n = 27). (B) To determine the amount of secretion remaining after the calcium ramp stimulation, a flash was applied 3 s after the ramp ended to increase calcium to high levels. The residual secretion in the CAPS DKO cells was larger than that of the DKO cells expressing open syntaxin and accounted for ∼65% of the total secretion, whereas the flash response in the open syntaxin–expressing CAPS DKO cells accounted for ∼25% of the total secretion. Error bars indicate mean ± SEM.

Mentions: Stimulation of mouse chromaffin cells with a slowly rising calcium concentration (calcium ramp) leads to biphasic secretion, the late phase of which is likely the result of priming during the stimulus, i.e., analogous to the sustained release during flash photolysis (Sørensen et al., 2002). In CAPS DKO cells, the late phase of secretion is either very small or absent (Liu et al., 2008). We examined the effects of ramp stimulation in DKO cells in which open syntaxin was expressed. In agreement with the data obtained by flash stimulation (Fig. 2), open syntaxin–expressing DKO cells showed strongly enhanced secretion (295.5 ± 38.9 fF; n = 29) as compared with untreated DKO cells (72.2 ± 13.2 fF; n = 27; Fig. 3 A). Secretion during ramp stimulation started slowly, so we used the second derivative of the capacitance trace to more accurately determine the increase in slope at the beginning of the secretory phase. The Ca2+ value at the time of a peak in the second derivative of the smoothed capacitance trace (Schonn et al., 2008), taken as the threshold Ca2+ concentration required for secretion, was ∼850 nM in both DKO and open syntaxin–expressing DKO cells. Both the response of DKO cells and that of the open syntaxin–expressing DKO cells were sigmoid. In spite of the greater secretion in the open syntaxin–expressing DKO cells (or perhaps as a result of this), secretion of the open syntaxin–expressing cells reached a plateau before the end of the stimulation. This was not the case in the DKO cells, which secreted throughout the stimulation.


Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

Liu Y, Schirra C, Edelmann L, Matti U, Rhee J, Hof D, Bruns D, Brose N, Rieger H, Stevens DR, Rettig J - J. Cell Biol. (2010)

The releasable pools in CAPS DKO cells expressing open syntaxin are rapidly exhausted. (A) The free calcium concentration (top) and the capacitance change (bottom) are shown. Those cells expressing open syntaxin exhibit very strong secretion (n = 29) compared with CAPS DKO cells not expressing open syntaxin (n = 27). (B) To determine the amount of secretion remaining after the calcium ramp stimulation, a flash was applied 3 s after the ramp ended to increase calcium to high levels. The residual secretion in the CAPS DKO cells was larger than that of the DKO cells expressing open syntaxin and accounted for ∼65% of the total secretion, whereas the flash response in the open syntaxin–expressing CAPS DKO cells accounted for ∼25% of the total secretion. Error bars indicate mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3101601&req=5

fig3: The releasable pools in CAPS DKO cells expressing open syntaxin are rapidly exhausted. (A) The free calcium concentration (top) and the capacitance change (bottom) are shown. Those cells expressing open syntaxin exhibit very strong secretion (n = 29) compared with CAPS DKO cells not expressing open syntaxin (n = 27). (B) To determine the amount of secretion remaining after the calcium ramp stimulation, a flash was applied 3 s after the ramp ended to increase calcium to high levels. The residual secretion in the CAPS DKO cells was larger than that of the DKO cells expressing open syntaxin and accounted for ∼65% of the total secretion, whereas the flash response in the open syntaxin–expressing CAPS DKO cells accounted for ∼25% of the total secretion. Error bars indicate mean ± SEM.
Mentions: Stimulation of mouse chromaffin cells with a slowly rising calcium concentration (calcium ramp) leads to biphasic secretion, the late phase of which is likely the result of priming during the stimulus, i.e., analogous to the sustained release during flash photolysis (Sørensen et al., 2002). In CAPS DKO cells, the late phase of secretion is either very small or absent (Liu et al., 2008). We examined the effects of ramp stimulation in DKO cells in which open syntaxin was expressed. In agreement with the data obtained by flash stimulation (Fig. 2), open syntaxin–expressing DKO cells showed strongly enhanced secretion (295.5 ± 38.9 fF; n = 29) as compared with untreated DKO cells (72.2 ± 13.2 fF; n = 27; Fig. 3 A). Secretion during ramp stimulation started slowly, so we used the second derivative of the capacitance trace to more accurately determine the increase in slope at the beginning of the secretory phase. The Ca2+ value at the time of a peak in the second derivative of the smoothed capacitance trace (Schonn et al., 2008), taken as the threshold Ca2+ concentration required for secretion, was ∼850 nM in both DKO and open syntaxin–expressing DKO cells. Both the response of DKO cells and that of the open syntaxin–expressing DKO cells were sigmoid. In spite of the greater secretion in the open syntaxin–expressing DKO cells (or perhaps as a result of this), secretion of the open syntaxin–expressing cells reached a plateau before the end of the stimulation. This was not the case in the DKO cells, which secreted throughout the stimulation.

Bottom Line: Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin.Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps.Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Physiologie, Universität des Saarlandes, 66421 Homburg, Germany.

ABSTRACT
Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

Show MeSH
Related in: MedlinePlus