Abstract
The kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+ were studied by capacitance (Cm) measurements with millisecond time resolution. After elevation of the internal Ca2+ concentration ([Ca2+]i), Cm rises rapidly with one or more exponentials. The time constant of the fastest component decreases for higher [Ca2+]i (range 3-600 microM) over three orders of magnitude before it saturates at approximately 1 ms. The corresponding maximal rates of secretion can be as fast as 100,000 fF/s or 40,000 vesicles/s. There is a Ca(2+)-dependent delay before Cm rises, which may reflect the kinetics of multiple Ca2+ ions binding to the secretory apparatus. The initial rise in Cm is described by models containing a sequence of two to four single Ca(2+)-binding steps followed by a rate-limiting exocytosis step. The predicted Ca2+ dissociation constant (Kd) of a single Ca(2+)-binding site is between 7 and 21 microM. At [Ca2+]i > 30 microM clear indications of a fast endocytotic process complicate the analysis of the secretory response.
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Selected References
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- Armstrong C. M. Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injection in squid axons. J Gen Physiol. 1969 Nov;54(5):553–575. doi: 10.1085/jgp.54.5.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Augustine G. J., Neher E. Calcium requirements for secretion in bovine chromaffin cells. J Physiol. 1992 May;450:247–271. doi: 10.1113/jphysiol.1992.sp019126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bittner G. D., Kennedy D. Quantitative aspects of transmitter release. J Cell Biol. 1970 Dec;47(3):585–592. doi: 10.1083/jcb.47.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bittner M. A., Holz R. W. Kinetic analysis of secretion from permeabilized adrenal chromaffin cells reveals distinct components. J Biol Chem. 1992 Aug 15;267(23):16219–16225. [PubMed] [Google Scholar]
- Brose N., Petrenko A. G., Südhof T. C., Jahn R. Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science. 1992 May 15;256(5059):1021–1025. doi: 10.1126/science.1589771. [DOI] [PubMed] [Google Scholar]
- Chow R. H., von Rüden L., Neher E. Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature. 1992 Mar 5;356(6364):60–63. doi: 10.1038/356060a0. [DOI] [PubMed] [Google Scholar]
- Cohen M. W., Jones O. T., Angelides K. J. Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin. J Neurosci. 1991 Apr;11(4):1032–1039. doi: 10.1523/JNEUROSCI.11-04-01032.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davletov B. A., Südhof T. C. A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding. J Biol Chem. 1993 Dec 15;268(35):26386–26390. [PubMed] [Google Scholar]
- Fenwick E. M., Marty A., Neher E. Sodium and calcium channels in bovine chromaffin cells. J Physiol. 1982 Oct;331:599–635. doi: 10.1113/jphysiol.1982.sp014394. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glenney J. R., Jr, Tack B., Powell M. A. Calpactins: two distinct Ca++-regulated phospholipid- and actin-binding proteins isolated from lung and placenta. J Cell Biol. 1987 Mar;104(3):503–511. doi: 10.1083/jcb.104.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
- Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
- Heinemann C., von Rüden L., Chow R. H., Neher E. A two-step model of secretion control in neuroendocrine cells. Pflugers Arch. 1993 Jul;424(2):105–112. doi: 10.1007/BF00374600. [DOI] [PubMed] [Google Scholar]
- Jahn R., Südhof T. C. Synaptic vesicles and exocytosis. Annu Rev Neurosci. 1994;17:219–246. doi: 10.1146/annurev.ne.17.030194.001251. [DOI] [PubMed] [Google Scholar]
- Jahromi S. S., Atwood H. L. Three-dimensional ultrastructure of the crayfish neuromuscular apparatus. J Cell Biol. 1974 Nov;63(2 Pt 1):599–613. doi: 10.1083/jcb.63.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KATZ B., MILEDI R. THE MEASUREMENT OF SYNAPTIC DELAY, AND THE TIME COURSE OF ACETYLCHOLINE RELEASE AT THE NEUROMUSCULAR JUNCTION. Proc R Soc Lond B Biol Sci. 1965 Feb 16;161:483–495. doi: 10.1098/rspb.1965.0016. [DOI] [PubMed] [Google Scholar]
- Kaplan J. H., Ellis-Davies G. C. Photolabile chelators for the rapid photorelease of divalent cations. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6571–6575. doi: 10.1073/pnas.85.17.6571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kawagoe K. T., Zimmerman J. B., Wightman R. M. Principles of voltammetry and microelectrode surface states. J Neurosci Methods. 1993 Jul;48(3):225–240. doi: 10.1016/0165-0270(93)90094-8. [DOI] [PubMed] [Google Scholar]
- Konishi M., Hollingworth S., Harkins A. B., Baylor S. M. Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra. J Gen Physiol. 1991 Feb;97(2):271–301. doi: 10.1085/jgp.97.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lindau M., Neher E. Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Arch. 1988 Feb;411(2):137–146. doi: 10.1007/BF00582306. [DOI] [PubMed] [Google Scholar]
- Neher E., Marty A. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6712–6716. doi: 10.1073/pnas.79.21.6712. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neher E., Zucker R. S. Multiple calcium-dependent processes related to secretion in bovine chromaffin cells. Neuron. 1993 Jan;10(1):21–30. doi: 10.1016/0896-6273(93)90238-m. [DOI] [PubMed] [Google Scholar]
- Nowycky M. C., Pinter M. J. Time courses of calcium and calcium-bound buffers following calcium influx in a model cell. Biophys J. 1993 Jan;64(1):77–91. doi: 10.1016/S0006-3495(93)81342-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parnas H., Hovav G., Parnas I. Effect of Ca2+ diffusion on the time course of neurotransmitter release. Biophys J. 1989 May;55(5):859–874. doi: 10.1016/S0006-3495(89)82885-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robitaille R., Adler E. M., Charlton M. P. Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron. 1990 Dec;5(6):773–779. doi: 10.1016/0896-6273(90)90336-e. [DOI] [PubMed] [Google Scholar]
- Thomas P., Lee A. K., Wong J. G., Almers W. A triggered mechanism retrieves membrane in seconds after Ca(2+)-stimulated exocytosis in single pituitary cells. J Cell Biol. 1994 Mar;124(5):667–675. doi: 10.1083/jcb.124.5.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P., Wong J. G., Almers W. Millisecond studies of secretion in single rat pituitary cells stimulated by flash photolysis of caged Ca2+. EMBO J. 1993 Jan;12(1):303–306. doi: 10.1002/j.1460-2075.1993.tb05657.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P., Wong J. G., Lee A. K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron. 1993 Jul;11(1):93–104. doi: 10.1016/0896-6273(93)90274-u. [DOI] [PubMed] [Google Scholar]
- Trifaró J. M., Vitale M. L. Cytoskeleton dynamics during neurotransmitter release. Trends Neurosci. 1993 Nov;16(11):466–472. doi: 10.1016/0166-2236(93)90079-2. [DOI] [PubMed] [Google Scholar]
- Wang W., Creutz C. E. Regulation of the chromaffin granule aggregating activity of annexin I by phosphorylation. Biochemistry. 1992 Oct 20;31(41):9934–9939. doi: 10.1021/bi00156a011. [DOI] [PubMed] [Google Scholar]
- Yamada W. M., Zucker R. S. Time course of transmitter release calculated from simulations of a calcium diffusion model. Biophys J. 1992 Mar;61(3):671–682. doi: 10.1016/S0006-3495(92)81872-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhou Z., Neher E. Mobile and immobile calcium buffers in bovine adrenal chromaffin cells. J Physiol. 1993 Sep;469:245–273. doi: 10.1113/jphysiol.1993.sp019813. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zucker R. S. Calcium and transmitter release. J Physiol Paris. 1993;87(1):25–36. doi: 10.1016/0928-4257(93)90021-k. [DOI] [PubMed] [Google Scholar]
- Zucker R. S. Changes in the statistics of transmitter release during facilitation. J Physiol. 1973 Mar;229(3):787–810. doi: 10.1113/jphysiol.1973.sp010167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zucker R. S. Effects of photolabile calcium chelators on fluorescent calcium indicators. Cell Calcium. 1992 Jan;13(1):29–40. doi: 10.1016/0143-4160(92)90027-p. [DOI] [PubMed] [Google Scholar]
- Zucker R. S. The calcium concentration clamp: spikes and reversible pulses using the photolabile chelator DM-nitrophen. Cell Calcium. 1993 Feb;14(2):87–100. doi: 10.1016/0143-4160(93)90079-l. [DOI] [PubMed] [Google Scholar]
- von Rüden L., Neher E. A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells. Science. 1993 Nov 12;262(5136):1061–1065. doi: 10.1126/science.8235626. [DOI] [PubMed] [Google Scholar]