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Local Electrostatic Interactions Determine the Diameter of Fusion Pores a
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a
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Alenka Guček , Jernej Jorgačevski , Urszula Górska , Boštjan Rituper , Marko Kreft Robert Zorec
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Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia b
Celica Biomedical, 1000 Ljubljana, Slovenia
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Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia Accepted author version posted online: 02 Apr 2015.
Click for updates To cite this article: Alenka Guček, Jernej Jorgačevski, Urszula Górska, Boštjan Rituper, Marko Kreft & Robert Zorec (2015): Local Electrostatic Interactions Determine the Diameter of Fusion Pores, Channels To link to this article: http://dx.doi.org/10.1080/19336950.2015.1007825
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1 Local Electrostatic Interactions Determine the Diameter of Fusion Pores Abbreviated title: Cations, HCN channels, fusion pore regulation Alenka Gu ek1, Jernej Jorga evski1,2, Urszula Górska1, Boštjan Rituper1, Marko Kreft1,2,3, Robert Zorec1,2 Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology,
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Celica Biomedical, 1000 Ljubljana, Slovenia
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Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia
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Address correspondence to: Professor Robert Zorec, E-mail:
[email protected]
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Article Addenda to: Calejo AI, Jorga evski J, Rituper B, Gu ek A, Pereira PM, Santos MA, Potokar M, Vardjan N, Kreft M, Gonçalves PP, Zorec R (2014) Hyperpolarization-Activated
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Cyclic Nucleotide-Gated Channels and cAMP-Dependent Modulation of Exocytosis in Cultured
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Rat Lactotrophs. J Neurosci 34:15638-15647
Keywords: HCN channels, lactotrophs, vesicles, astrocytes, exocytosis, fusion pore
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Abstract
In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response
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to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least two mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering
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Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
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local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were 1
2 detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore
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membrane composition.
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Introduction The merger between the vesicle and the plasma membranes is present in the majority of eukaryotic cells in the form of constitutive exocytosis1. Specialized cells, like neurons and
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neuroendocrine cells, additionally exhibit regulated exocytosis, triggered by a physiologic
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merger leads to the formation of a fusion pore, a water-filled channel, which may eventually
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reversibly close (transient exocytosis) or fully widen, allowing the integration of the vesicle membrane with the plasma membrane (full-fusion exocytosis)4, 5. A large body of evidence
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suggests that the transition from transient fusion, where the fusion pore diameter fluctuates between a wide open and a virtually closed state, to a fully-fused state, is the rate limiting
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process6-8. A fusion pore is formed by deforming membranes into highly curved regions and the
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fact that they can persist in this apparently energetically unfavourable state9 indicates that certain stabilization factors are likely present.
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The pivotal contribution from an energetics standpoint, especially in the early stages of the fusion pore formation, is likely provided by the SNARE (Soluble N-ethylmaleimide sensitive
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factor attachment protein receptor) complex formation10. Nevertheless, several other SNAREbinding proteins, be they individual or in complexes, influence the formation and the expansion of fusion pores; including synaptotagmins11, complexins12, and Sec1/Munc18 (SM) proteins13.
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stimulus2, usually in the form of increased free intracellular calcium ([Ca2+]i)3. Membrane
Lipids also affect the fusibility of lipid bilayers14 and can regulate the fusion process by manipulating the SNARE complex15. In addition to specific interactions, membrane constituents
(lipids, proteins or their complexes) can affect the energy landscape via their shape. If their shape is non-axisymmetric (anisotropic), membrane constituents can stabilize the highly curved fusion 3
4 pore16, similarly as voussoirs (wedge-shaped stones) in a Roman arch. Moreover, membrane constituents usually contain one or more polar groups, which may be ionized17. The accumulation of ions at polarized membrane regions may affect local curvature due to lipid demixing. If such a region is associated with the fusion pore, then the accumulation of ions (at
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the cytoplasmic or extracellular side) could affect the stability and local curvature of the pore, as
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Recently, we reported that Hyperpolarization-activated Cyclic Nucleotide-gated (HCN)
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channels modulate fusion pore properties19. HCN channels are permeable to cations (Na+, K+ and Ca2+)20-22 and likely affect exocytosis indirectly by increasing the local [Ca2+]i, but may also
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contribute directly via electrostatic interactions with charged membrane constituents near the fusion pore. Here, we assessed the contribution of electrostatic interactions, mediated by
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extracellular di- and trivalent cations, to changes in fusion pore conductance, a parameter reporting pore geometry and in particular fusion pore diameter23. For this we have first modified
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the extracellular concentration of cations by removing Ca2+ from or by adding Al3+ to the
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extracellular solution and then studied the discrete states of fusion pore. Results and discussion
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HCN2 channels modulate exocytosis It was shown previously that an increase in intracellular second messenger cAMP affects exocytotic events in cultured pituitary lactotrophs19 and that some of the modulations are
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proposed by Kabaso et al.18.
mediated by HCN channels, which are expressed in the plasma membrane and in the membrane of secretory vesicles24. If the plasma membrane-resident HCN channels are activated, then this may increase the local [Ca2+]i, a stimulus known to increase the exocytotic activity25. However, in lactotrophs overexpressing HCN2 channels, the overall [Ca2+]i was lower compared to non-
5 transfected lactotrophs and an increase in intracellular cAMP did not significantly affect [Ca2+]i24, consistent with previous results26. A possible explanation is that the activation of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)27 decreased global [Ca2+]i, as depicted in the model (Fig. 1). How, then, did exocytotic activity increase in the study by Calejo et al.24? It is
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possible that Ca2+ is still increased locally, in the proximity of the fusion pore. Alternatively,
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charged ions that may potentially lead to lipid demixing at the region of the fusion pore18. To
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date, the exact proteo-lipidic composition of the fusion pore remains unclear28. However, it is safe to assume that anisotropic membrane constituents (proteins, lipids or other nanodomains)
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can attribute to its stability29, 30. The constituents of biological membranes (lipids, glycoproteins, glycolipids, etc.) frequently carry one or more ionized or polar groups17 and are influenced by
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local cation clouds31, 32. Prime candidates are widespread anisotropic anionic lipids, known to bind di- and trivalent cations, such as phosphoinositides (PI), phosphatidic acid (PA) and,
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particularly, phosphatidylserine (PS)33. The interaction of anionic lipids with cations (especially
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Ca2+) can dehydrate anionic lipid head groups and consequently alter local membrane curvature and lipid molecule packing into the local membrane regions (e.g. in the fusion pore region)32,
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leading to lateral phase separation of membrane components34. These changes can then affect anisotropic neutral (e.g. cholesterol) and zwitterionic (e.g. phosphatidylethanolamine) lipids33, known to participate in regulated exocytosis35. Therefore, changes in cation concentration in the
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HCN channels could modulate exocytosis through a more general local cloud of positively
pore area likely influence the membrane fusion process during exocytosis. Removal of extracellular divalent cations results in fusion pores with relatively wide diameters HCN2 channels have been shown to reside in or near vesicles in lactotrophs24. Manipulation of 5
6 HCN channels, where their presence was either increased by HCN2 overexpression or their rhythmic re-opening was accelerated by cAMP, amplifies HCN-specific Ih current24, which likely increases local cytoplasmic cation concentration near fusion pores (Fig. 1A). Simultaneously, the proportion of narrow fusion pores was decreased24. To assess if the observed
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effect may in part be attributed to the electrostatic interactions, we designed a conceptually
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removed Ca2+ ions from the extracellular space. Then, cell-attached patch-clamp technique was
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employed to measure reversible discrete steps in the membrane capacitance (Cm), corresponding to unitary, transient fusion events of vesicles with the plasma membrane in real time36.
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To test the robustness of our predictions on the general importance of electrostatic interactions in regulated exocytosis, we performed these experiments on a different cell type – astrocytes.
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Astrocytes are electrically silent and abundant glial cells in the brain, which actively contribute to information processing in the central nervous system by releasing gliotransmitters37. In
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astrocytes, reversible discrete steps in Cm were observed in controls with 2 mM Ca2+ (Fig. 2A)
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and in conditions without Ca2+ (Fig. 2B). Here, we focused only on reversible exocytotic events, which likely represent transient fusion pore openings38. A fraction of reversible events exhibit a
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measurable (narrow) fusion pore conductance, which is discerned by the projection between the imaginary (Im) and the real (Re) parts of admittance signals29. In controls half of the reversible events exhibited projections to the Re trace (Fig. 2A). For these events the average fusion pore
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similar experiment, where instead of increasing divalent cation concentration intracellularly, we
conductance of 35 ± 4 pS was calculated, which corresponds to the average fusion pore diameter of 0.73 ± 0.05 nm (n = 12 cells) (see Materials and Methods for details). In contrast, in astrocytes that were bathed in Ca2+-free ECS, reversible exocytotic events exhibited no projections to the Re trace (Fig 2B), indicating fusion pores with relatively wide diameters. Experimentally
7 determined detection limit for projected exocytotic events with our recording system was determined at ~2.6 nm. Non-projected exocytotic events therefore exhibit fusion pores wider than ~2.6 nm in diameter. Moreover, the frequency of all reversible exocytotic events was
with 2 mM Ca2+ (2.2 ± 0.2 events/min, n = 12 cells, P < 0.001, U-test).
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significantly lower in Ca2+-free ECS (0.14 ± 0.06 events/min, n = 12 cells) compared to ECS
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However, fusion pores that are formed have relatively wide diameters, which is in line with the
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proposed model on Fig. 1A. Moreover, compared to HCN2 overexpressing cells, where [Ca2+]i was also shown to be reduced24, the observed effect was even stronger, since none of the events
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exhibited projected (narrow) fusion pore (compared to the 17% of narrow fusion pores observed
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in cells overexpressing HCN2).
Extracellular trivalent cations stabilize fusion pores with relatively narrow diameters In lactotrophs exposed to HCN2 blocker (ZD7288), the HCN-specific Ih current was decreased,
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indicating a reduction in local cytoplasmic cation concentration (Fig. 1B)24. In this case the
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proportion of narrow fusion pores recorded was increased24. This effect was even more profound after the addition of cAMP, which likely triggered the activation of SERCA pumps, subsequently
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decreasing cytoplasmic cation (Ca2+) concentration24. To further validate our model, we conducted conceptually the opposite experiments, as depicted in model B (Fig. 1). To increase the local cation concentration in the extracellular space, we monitored discrete changes in Cm of
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This experiment shows that the removal of extracellular Ca2+ reduces exocytotic activity.
lactotrophs bathed in ECS containing 30 µM Al3+. Here, the majority of reversible exocytotic events were projected to the Re trace of the admittance signal (Fig. 3A). Compared to the previous reports, where ~25% of reversible events exhibited projections to the Re trace in conditions where normal ECS was used24, Al3+-treatment 7
8 significantly increased the percentage of reversible events to 77% (Fig. 3B), suggesting strong stabilization of narrow exocytotic fusion pores (Fig. 3). Although Al3+ has a wide range of modus operandi39, electrostatic interactions could, as proposed in the model (Fig. 1B), be responsible for this outcome. The average frequency of all reversible exocytotic events was
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significantly lower in Al3+ treated lactotrophs (0.45 ± 0.09 events/min, n = 8 cells) compared to
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Local cation concentration modulates discrete fusion pore state
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We propose that changes in divalent cation concentration near fusion pores determines the extent of cation binding with charged membrane constituents, which affects membrane curvature and
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affects lipid demixing. These changes then, in turn, provide a framework responsible for the stabilization of fusion pore configurations, as summarized in Fig. 4. This is consistent with
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results acquired on chromaffin cells where an increase in extracellular calcium concentration shifts the mode of exocytosis to kiss-and run41 and reduces the quantum content of a single
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exocytotic event42. The results presented in this work show a role of electrostatic interactions in
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affecting the transitions between discrete states of fusion pores. However, they do not argue against the necessity of protein-protein interactions (such as the formation of the SNARE
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complex) in this process.
In summary, our results show that changes in the extracellular concentration of cations directly modulate fusion pore conductance, a parameter related to the fusion pore diameter. We propose
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controls (2.5 ± 0.9 events/min, n = 8 cells; P < 0.001, U-test), as previously reported40.
that the fusion pore stability in either a wide or a narrow configuration is affected by electrostatic interactions mediated by cations adjacent to the fusion pore. These observations bear physiological significance, since extracellular calcium concentration is reduced during activity in the nervous system, which may regulate synaptic activity via sensing extracellular Ca2+ via
9 GPCR receptors43 and as shown in this study by directly affecting the fusion pore properties as well. Materials and Methods Materials and solutions
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Extracellular solution (ECS) for astrocytes contained (in mM): 130 NaCl, 5 KCl, 2 CaCl2, 1
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Ca2+, CaCl2 was replaced with NaCl. Extracellular solution used for lactotrophs contained (in
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mM): 130 NaCl, 5 KCl, 8 CaCl2, 1 MgCl2, 10 D-glucose, 10 HEPES (N-2Hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and pH 7.2 (with NaOH). AlCl3 was prepared
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as a stock solution and was added to the growth medium and ECS at 30 µM (final
Sigma, unless stated otherwise. Cell Cultures
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concentration). Osmolarity of solutions was ~300 mOsm. All chemicals were purchased from
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Astrocytes were isolated from cortices of 2-3-day-old Wistar rats36 and maintained in growth
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medium (high-glucose Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum (FBS), 1 mM pyruvate, 2 mM glutamine and 25 µg/ml penicillin/streptomycin at 37 °C,
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95% air/5% CO2). Confluent cultures were shaken overnight (225 rpm); the medium was changed the next morning and the process was repeated three times. After the third shaking, the cells were trypsinized and cultured in flat culture tubes until confluence.
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MgCl2, 10 D-glucose, 10 HEPES and pH 7.2 (with NaOH). In experiments without extracellular
Lactotroph primary cultures were prepared as described36. After isolation, lactotrophs were plated onto poly-L-lysine coated glass coverslips and kept in high-glucose Dulbecco’s modified
Eagle’s medium (Invitrogen) with 10% newborn calf serum and L-glutamine at 37 °C with 95% humidity and 5% CO2. 9
10 All experiments were performed within a period of one to three days after cell isolation. Animals were euthanized according to the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and Animal Protection Act (Official Gazette of the RS, No. 38/13). The
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protocol for the euthanization of the animals used in our study was approved by the Veterinary
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34401-29/2009/2).
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Electrophysiologic measurements of Cm
Cell-attached membrane capacitance measurements (Cm) were performed with a dual-phase lock-
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in patch-clamp amplifier (SWAM IIC, Celica, Ljubljana, Slovenia) as described36 at room temperature. We used fire-polished pipettes, heavily coated with Sylgard (to reduce stray
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capacitance), and with the resistance of 2–5 MΩ. The bath and pipettes contained ECS. A sine
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wave (111 mV rms and 1591 Hz for lactotrophs or 6364 Hz for astrocytes) was applied to the pipette and the holding steady state pipette potential was held at 0 mV. During the experiments,
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the phase angle was adjusted to nullify the changes in the real (Re) trace in response to the manually generated 10 fF calibration pulses.
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Data Analysis
We used custom-made MATLAB (Math Works, Natick, MA, USA) subroutine (CellAn, Celica, Slovenia) to analyze exocytotic events. Fusion event was considered detectable, if the signal-to-
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Administration of the Ministry for Agriculture and the Environment of the RS (permit No:
noise ratio exceeded 3:1. We analysed only reversible exocytotic events, where an off-step in Im followed an on-step within 3 s. Vesicle diameter was assessed for all exocytotic events by determining vesicle capacitance (Cv): Cv = (Re2 + Im2)/Im/ω, where Im denotes the amplitude
change in the imaginary part of the admittance trace, Re is the amplitude change in the real part
11 of the admittance trace and ω is the angular frequency44. Vesicle diameter was calculated assuming spherical geometry and using a specific membrane capacitance of 8 fF/µm2 (lactotrophs)3 and of 10 fF/µm2 (astrocytes)45. For transient exocytotic events in the Im that exhibited measurable projections to the Re, we calculated fusion pore conductance (Gp): Gp =
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(Re2 + Im2)/Re. Gp was used to estimate the fusion-pore radius by using the equation Gp =
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Ωcm) and λ is the estimated length of a gap junction channel (15 nm)46.
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SigmaPlot was used for the statistical analyses. Values are presented as mean ± SEM.
(*), P < 0.01 (**) and P < 0.001 (***). Funding
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Differences between samples were tested with the Mann-Whitney U-test, considering P < 0.05
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This work was supported by the Slovenian Research Agency grants: P3 310, J3 6790, J3 4051,
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J3 4146, L3 3654; J3 3236, CIPKEBIP, COST Nanonet). Disclosure
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The funders had no role in study design, data collection and analysis, decision to publish, or
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preparation of the manuscript. The authors declare no competing financial interests.
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(πr2)/(ρλ), where r denotes the fusion-pore radius, ρ is the estimated resistivity of the saline (100
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Figure 1. A model demonstrating how HCN channels affect fusion of secretory vesicles.
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HCN channel activation in the plasma membrane and/or in the membrane of fused vesicles increases the intracellular concentration of cations (due to their established role in the process of
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exocytosis, only calcium ions are drawn) in the close proximity of the fusion pore. cAMP directly facilitates the opening of HCN channels, however, cAMP can also activate
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sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) through protein kinase A - dependent
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mechanism, by reducing the association between SERCA and its inhibitor phospholamban in some cell types47. As a consequence, HCN channels may promote a local decrease in extracellular cation concentration. Increased intracellular cation levels affect fusion pore
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properties either through SNARE complex or by electrostatic interactions as proposed by Kabaso et al.18. Low extracellular divalent cation concentration promotes the formation of wide fusion
pores (A) and high extracellular divalent cation concentration supports the formation of narrow fusion pores (B). Grey areas denote the intracellular domain adjacent to a fusion pore. 17
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Figure 2. Calcium removal from ECS results in wide fusion pores in astrocytes. (A) Representative discrete steps in membrane capacitance (Cm) denote transient fusion
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exocytotic events. The top trace shows the real (Re) part and the bottom one the imaginary (Im) part of the admittance signal in controls bathed in extracellular solution (ECS) containing 2mM
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Ca2+ (ECS with Ca2+). The Im trace exhibits two types of reversible exocytotic events: those without projections (left) and those with projections to the Re trace (right). In controls, overall,
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50% of reversible events in the Im trace exhibited projections to the Re trace. (B) Representative
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discrete steps in Cm from astrocytes bathed in Ca2+-free ECS (ECS without Ca2+). No reversible events, detected in the Im trace, exhibited a projection to the Re trace, indicating wide diameter fusion pores. Asterisks denote truncated calibration pulses.
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Representative discrete steps in membrane capacitance (Cm) in lactotrophs bathed in 30 µM Al3+.
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The top trace shows the real (Re) part and the bottom trace shows the imaginary (Im;
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proportional to Cm) part of the admittance signals. Two representative transient fusion events with projections to the Re trace are shown. Previous reports indicate that ~25% of reversible
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events are projected to the Re trace in Al3+-free ECS24. When lactotrophs were bathed in Al3+, we detected 77% projected events, indicating that most transient exocytotic events exhibited only
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narrow fusion pores.
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Figure 3. Incubation in Al3+-enriched ECS results in narrow fusion pores in lactotrophs.
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Figure 4. Summary of electrostatic modulation due to the presence/absence of extracellular
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cations (di- and trivalent) on fusion pore conductance (i.e. pore diameter).
From the previously published data by Calejo et al.24 and from the new data in this paper, we
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conclude that fusion pore conductance (a parameter related to pore morphology) is modulated by cations adjacent to the fusion pore. A reduction of extracellular cation concentration (or an
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increase of intracellular cation concentration) stabilizes fusion pore in more conductive, wider configuration. Conversely, the increase of extracellular cation concentration (or a decrease of
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intracellular cation concentration) stabilizes fusion pores in less conductive, narrower
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configuration. (Control; HCN2, HCN2 transfected cells; HCN/dbcAMP, HCN2 transfected dbcAMP treated cells; noCa2+, ECS without Ca2+; ZD, ZD7288 treated cells; ZD/dbcAMP,
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ZD7288 and dbcAMP treated cells). Numbers indicate the fraction of exocytotic events (in %) with narrow fusion pores compared to all observed events.
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