Fast Exocytosis with Few Ca2+ Channels in Insulin-Secreting Mouse Pancreatic B Cells
Open accessAbstract
The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.
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The maximal Ca2+ current amplitude in isolated mouse β-cells is ≈100 pA, and the number of voltage-gated Ca2+ channels per β-cell has been estimated as ~500 by non-stationary fluctuation analysis.
“The maximal Ca2+ current amplitude in isolated mouse β-cells is ≈100 pA, and the number of voltage-gated Ca2+ channels per β-cell has been estimated as ~500 by non-stationary fluctuation analysis (48).”
From Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men (2018) · cites this paper as [48]
The section “The B cell contains <500 α1C L-type Ca2+ channels” estimates 450 channels per cell, and Electrophysiology specifies that the estimate used nonstationary fluctuation analysis. The same section gives the largest Ca2+ current as approximately 80 pA, rather than approximately 100 pA. Page numbers are not available in the supplied passages. (LLM, conf=0.85)
Experimental — accuracy not yet measuredCa2+ channels are not evenly distributed in β-cells and aggregate into clusters that associate with the secretory granules.
“However, it turns out that the Ca2+ channels are not evenly distributed in the β-cells and that they aggregate into clusters that associate with the secretory granules: single-channel recordings revealed that whereas most membrane patches contain no active Ca2+ channels, other patches contained 3 or more active channels, suggesting that their distribution is non-stochastic (48).”
From Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men (2018) · cites this paper as [48]
In “Evidence for Ca²⁺ channel clustering” (page unavailable), more than 60% of patches had no active channels, while four of seven patches with channel activity had three simultaneously active channels. “Estimation of the Ca²⁺ channel: granule stoichiometry” concludes that channels are not randomly distributed, but describes their association with individual secretory granules as speculation rather than an established finding. (LLM, conf=0.88)
Experimental — accuracy not yet measuredExocytosis evoked by a train of depolarizations or photorelease of caged Ca2+, two conditions that can be assumed to produce a high [Ca2+]i throughout the β-cell, was unaffected by the synprint peptide.
“Exocytosis evoked by a train of depolarizations (48) or photorelease of caged Ca2+ (731), two conditions that can be assumed to produce a high [Ca2+]I throughout the β-cell, was unaffected by the synprint peptide.”
From Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men (2018) · cites this paper as [48]
In “Assembly of a functional complex between Ca²⁺ channels and secretory granules” (page unavailable), the paper reports that later pulses in a depolarization train evoked responses comparable to controls despite the synprint peptide, but the first response was much reduced. The “Solutions” section (page unavailable) specifies the peptide in solution I and caged-Ca²⁺ photorelease in solution II; the copy does not report that photorelease-evoked exocytosis was unaffected by the peptide. (LLM, conf=0.88)
Experimental — accuracy not yet measuredClustering of Ca2+ channels has dramatic effects on the likelihood of a granule being exposed to elevated Ca2+ for long enough (10 ms) to evoke exocytosis: from 0.5% for individual Ca2+ channels to ~20% for a triplet of Ca2+ channels.
“Clustering of Ca2+ channels has, due to spatiotemporal [Ca2+]i domain overlap (Figure 22B-C), dramatic effects on the likelihood of a granule being exposed to elevated Ca2+ for long enough (10 ms) to evoke exocytosis: from 0.5% for individual Ca2+ channels to ~20% for a triplet of Ca2+ channels (48).”
From Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men (2018) · cites this paper as [48]
Based on the length of the synprint peptide, the secretory granules must reside within 10 nm from the inner mouth of the Ca2+ channel.
“Based on the length of the synprint peptide, it has been estimated that the secretory granules must reside within 10 nm from the inner mouth of the Ca2+ channel (48) (Figure 20A).”
From Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men (2018) · cites this paper as [48]
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Cites
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- Authors
- Barg, S., Ma, X., Eliasson, L., Galvanovskis, J., Göpel, S. O., Obermüller, S., Platzer, J., Renström, E., Trus, M., Atlas, D., Striessnig, J., & Rorsman, P.
- Affiliations
- Lund University (SE)
- Universität Innsbruck (AT)
- Hebrew University of Jerusalem (IL)
- Corresponding author
- Rorsman, Patrik
- Venue
- Biophysical Journal
- Publisher
- Elsevier BV
- Published
- 2001-12
- Volume
- 81
- Issue
- 6
- Pages
- 3308-3323
- ISSN
- 0006-3495 (ISSN-L)
- PMC
- PMC1301788
- OpenAlex
- W2046901610
- Cited by
- 255
- References
- 2
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 4 · 2023: 3 · 2022: 7 · 2021: 13 · 2020: 7 · 2019: 9 · 2018: 2 · 2017: 9 · 2016: 11 · 2015: 13 · 2014: 15 · 2013: 4 · 2012: 14
- FWCI
- 13.57
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Biophysical Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Lund University Publications (Lund University) (submittedVersion)
- Funders
- Knut och Alice Wallenbergs Stiftelse
- Medicinska Forskningsrådet (12234, 13147, 13509, 8647)
- European Association for the Study of Diabetes
- Novo Nordisk Fonden
- European Commission (HPRN-CT-2000-00082)
- Juvenile Diabetes Research Foundation International
- Topics
- Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
- Keywords
- insulin secretion, pancreatic beta cells, L-type calcium channels, exocytosis, secretory granules, readily releasable pool, alpha1C subunit, cytoplasmic loop, Bay K 8644, single-channel activity
- MeSH terms
- Animals, B-Lymphocytes, Calcium, Cell Membrane, Electrophysiology, Exocytosis, Insulin, Microscopy, Fluorescence, Pancreas, Protein Binding, Time Factors, Calcium Channels, Mice, Knockout, Reverse Transcriptase Polymerase Chain Reaction, Mice
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