Changes In Membrane Capacitance Research Articles

“Clip Detecting” with Series Resistance Compensation using an Automated Patch Clamp System

One of the significant challenges for automated patch clamp has been to incorporate amplifiers with the ability of adjusting to changes in membrane capacitance, series resistance (Rs) and voltage-clamp time constants without user intervention. If these adjustments are not made correctly there is a risk of generating un-precise data or even losing data due to oscillations in the recorded trace. Rs compensation capability of the patch clamp amplifier of QPatch is presented. A new “clip detector feature” allowing Rs to be temporarily turned off avoiding loss of cells due to fatal oscillations, and thereby increasing the data throughput while maintaining high quality recordings, is presented.

Exocytosis in the Frog Amphibian Papilla

Using whole-cell patch-clamp recordings, we measured changes in membrane capacitance (ΔC (m)) in two subsets of hair cells from the leopard frog amphibian papilla (AP): the low-frequency (100-500 Hz), rostral hair cells and the high-frequency (500-1200 Hz), caudal hair cells, in order to investigate tonotopic differences in exocytosis. Depolarizations of both rostral and caudal hair cells evoked robust ΔC (m) responses of similar amplitude. However, the calcium dependence of release, i.e., the relationship between ΔC (m) relative to the amount of calcium influx (Q (Ca) (2+)), was found to be linear in rostral hair cells but supra-linear in caudal hair cells. In addition, the higher numbers of vesicles released at caudal hair cell active zones suggests increased temporal precision of caudal hair cell exocytosis. ΔC (m) responses were also obtained in response to sinusoidal stimuli of varying frequency, but neither rostral nor caudal hair cell ΔC (m) revealed any frequency selectivity. While all AP hair cells express both otoferlin and synaptotagmin IV (SytIV), we obtained evidence of a tonotopic distribution of the calcium buffer calretinin which may further increase temporal resolution at the level of the hair cell synapse. Our findings suggest that the low (rostral) and high (caudal) frequency hair cells apply different mechanisms for fine-tuning exocytosis.

Developmental Acquisition of a Rapid Calcium-Regulated Vesicle Supply Allows Sustained High Rates of Exocytosis in Auditory Hair Cells

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca2+ currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca2+ efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca2+ sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca2+-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane.

Myosin 5 is involved in cAMP‐induced ENaC trafficking in a mCCD cell line

The molecular motor which traffics epithelial sodium channels (ENaC) to and from the apical membrane is still unknown. We hypothesized that myosins, specifically Myosin 5 (Myo5), is involved in ENaC exocytosis. To determine the role of Myo5 in ENaC trafficking, we knocked down Myo5a and Myo5c in mCCD cells using shRNAs. We then measured the short-circuit current (Isc) and membrane capacitance change (ΔCT) of cells in Ussing chambers before and following cAMP activation. The Isc and CT change following cAMP-induced trafficking in knockdowns was significantly smaller than mCCD controls. Cells with Myo5c knocked down had a ΔIsc of 2.9 μA ± 1.03 μA compared to controls ΔIsc of 4.7 μA ± 0.68μA. There was a significant reduction in ΔCT (21% for controls vs. 14% in Myo5c) following cAMP stimulation. No significant change in Isc or CT response was observed for Myo5a knockdowns. There was no significant difference in basal (unstimulated) Isc between controls and Myo5c knockdowns. These results suggest that Myo5c is specifically involved in cAMP-induced ENaC trafficking to the apical membrane in response to cAMP stimulation and not constitutive ENaC protein turnover.

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3− secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

Besides being a Ca²-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl, HCO₃, and Na, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO₃- secretion. CFTR served as the principal route for PTH-stimulated apical HCOV efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCOV was probably supplied by basolateral uptake via the electrogenic Na⁺-HCO₃⁻ cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H⁺ might be extruded by both apical and basolateral Na/H exchangers. Furthermore, the PTH-stimulated HCO₃-secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14-22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca²⁺ chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO₃- secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.

Regulation of Calcium Channels and Exocytosis in Mouse Adrenal Chromaffin Cells by Prostaglandin EP3 Receptors

Prostaglandin (PG) E(2) controls numerous physiological functions through a family of cognate G protein-coupled receptors (EP1-EP4). Targeting specific EP receptors might be therapeutically useful and reduce side effects associated with nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors that block prostanoid synthesis. Systemic immune challenge and inflammatory cytokines have been shown to increase expression of the synthetic enzymes for PGE(2) in the adrenal gland. Catecholamines and other hormones, released from adrenal chromaffin cells in response to Ca(2+) influx through voltage-gated Ca(2+) channels, play central roles in homeostatic function and the coordinated stress response. However, long-term elevation of circulating catecholamines contributes to the pathogenesis of hypertension and heart failure. Here, we investigated the EP receptor(s) and cellular mechanisms by which PGE(2) might modulate chromaffin cell function. PGE(2) did not alter resting intracellular [Ca(2+)] or the peak amplitude of nicotinic acetylcholine receptor currents, but it did inhibit Ca(V)2 voltage-gated Ca(2+) channel currents (I(Ca)). This inhibition was voltage-dependent and mediated by pertussis toxin-sensitive G proteins, consistent with a direct Gβγ subunit-mediated mechanism common to other G(i/o)-coupled receptors. mRNA for all four EP receptors was detected, but using selective pharmacological tools and EP receptor knockout mice, we demonstrated that EP3 receptors mediate the inhibition of I(Ca). Finally, changes in membrane capacitance showed that Ca(2+)-dependent exocytosis was reduced in parallel with I(Ca). To our knowledge, this is the first study of EP receptor signaling in mouse chromaffin cells and identifies a molecular mechanism for paracrine regulation of neuroendocrine function by PGE(2).

Phase Association and Binding Energetics of Swcnts into Phospholipid Langmuir Monolayers

Single-walled carbon nanotubes (SWCNTs) have been identified as promising candidates for targeted drug delivery due to their low toxicity and ability to be functionalized using various bioactive groups. It is currently undetermined what mechanical and biological mechanism(s) are responsible for uptake into cells. We have used dispersed pristine SWCNTs to study mechanisms responsible for cellular uptake using cellular systems and in vitro model membrane systems. Fluorescence lifetime imaging microscopy (FLIM) of RhoB-GFP, a marker of endocytosis, shows functional spatial and temporal localization of endosomes with SWCNTs. Results demonstrate an increase in the number of endosomes after SWCNTs exposure indicating a stimulation of endocytosis. Measurements of SWCNT on artificial tethered bilayer membrane systems have shown no change in membrane capacitance indicating no penetration or disruption of membranes by SWCNTs. Preliminary results with Langmuir monolayers of lipids exposed to SWCNTs shows no significant increase in surface pressure from the control also indicating no significant uptake of SWCNTs. In sum, results support cellular mediated endocytosis of SWCNTs as the preferred mechanism for cellular uptake.

CaV2.1 (P/Q channel) interaction with synaptic proteins is essential for depolarization-evoked release

It is well established that syntaxin 1A (Sx1A), SNAP-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca2+ channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while Sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca2+-independent coupling of the human neuronal P/Q-type calcium channel (CaV2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of CaV2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by CaV1.2, CaV2.2, and CaV2.3. The distinct kinetic properties of CaV2.1 acquired by its close association with Syt1 and t-SNAREs suggest that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca2+. To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BAPTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted CaV2.1 release is consistent with the model in which the VGCC acts from within the exocytotic complex playing a signaling role in triggering release. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca2+ entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential.

Permissive role of sphingosine on calcium-dependent endocytosis in chromaffin cells

Sphingosine has been shown to modulate neurotransmitter release. Because membrane fusion and fission involve lipid metabolism, we asked here whether sphingosine had a role in regulating endocytosis. To explore this hypothesis, we monitored changes of membrane capacitance (Cm) to study the effects of intracellular sphingosine on membrane retrieval after chromaffin cell stimulation with depolarising pulses (DPs). We found that: (1) sphingosine dialysis through the patch-clamp pipette (SpD) using the whole-cell configuration of the patch-clamp technique (WCC) favours the appearance of a pronounced endocytotic response; (2) SpD-elicited endocytosis was Ca(2+)-dependent but Ba(2+) did not substitute Ca(2+); (3) under WCC, such endocytotic response disappeared with repetitive DPs; (4) in cells preincubated with sphingomyelinase to augment endogenous sphingosine synthesis, and then voltage-clamped under the perforated-patch configuration of the patch-clamp technique (PPC), endocytosis decayed little with repeated stimulation; (5) sphingosine-1-phosphate (S1P), a metabolite of sphingosine, had a meagre effect on endocytosis; and (6) neither dynamin inhibitor dynasore nor calmodulin blocker calmidazolium affected the sphingosine elicited endocytosis. We believe this is the first report showing that sphingosine plays a permissive role in activating Ca(2+)-dependent endocytosis during cell depolarisation. This effect requires high subplasmalemmal cytosolic Ca(2+) concentrations and a cytosolic factor(s) that is dialysed with the pipette solution. Independence of dynamin and calmodulin suggests that sphingosine-dependent endocytosis could be a novel, more direct pathway for vesicle recycling under mild depolarisation stimuli.

SV2 Acts via Presynaptic Calcium to Regulate Neurotransmitter Release

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

Characterisation of bipolar cell synaptic transmission in goldfish retina using paired recordings

Direct recordings from the large axon terminals of goldfish retinal bipolar cells (BCs) have revealed detailed information about the properties and regulation of exocytosis at this ribbon-type synapse. However, the relationship between BC exocytosis and evoked postsynaptic responses in amacrine and ganglion cells is not known. To address this, I have made paired recordings from BC terminals (BCTs) and neurons in the ganglion cell layer (GCL) in goldfish retinal slices. BCT depolarisation evoked short-latency, AMPA/kainate receptor-mediated EPSCs in connected GCL neurons. NMDA receptors contributed to the response at +40 mV but not at 60 mV. Evoked EPSCs contained multiple temporal components that differed in their relative amplitudes between pairs. Changing the duration or amplitude of the presynaptic stimulus affected the size and kinetics of the EPSC, with weaker stimuli slowing the EPSC activation rate. Paired-pulse stimulation caused greater depression of fast than slow EPSC components. A linear relationship was found between the amount of BCT exocytosis, measured via changes in membrane capacitance, and the charge of evoked EPSCs, whether they were mediated by AMPA/kainate receptors alone or in combination with NMDA receptors. In addition, analysis of miniature EPSCs in GCL neurons provided estimates of the quantal content of evoked EPSCs. The results demonstrate the feasibility of using this paired recording system to study synaptic transfer at ribbon synapses, and indicate that both the rapid and sustained phases of BC exocytosis are encoded in the postsynaptic response.

Involvement of Ca2+Channel Synprint Site in Synaptic Vesicle Endocytosis

The synaptic protein interaction (synprint) site of the voltage-gated Ca(2+) channel (VGCC) alpha1 subunit can interact with proteins involved in exocytosis, and it is therefore thought to be essential for exocytosis of synaptic vesicles. Here we report that the synprint site can also directly bind the mu subunit of AP-2, an adaptor protein for clathrin-mediated endocytosis, in competition with the synaptotagmin 1 (Syt 1) C2B domain. In brain lysates, the AP-2-synprint interaction occurred over a wide range of Ca(2+) concentrations but was inhibited at high Ca(2+) concentrations, in which Syt 1 interacted with synprint site. At the calyx of Held synapse in rat brainstem slices, direct presynaptic loading of the synprint fragment peptide blocked endocytic, but not exocytic, membrane capacitance changes. We propose that the VGCC synprint site is involved in synaptic vesicle endocytosis, rather than exocytosis, in the nerve terminal, via Ca(2+)-dependent interactions with AP-2 and Syt.

Effects of Rab3 and Rab27 on Exocytotic Activity and Docking in Mouse Chromaffin Cells

The small GTPases Rab3A and Rab27A are endogenously expressed and specifically localized to dense-core vesicles in PC12 cells and are implicated in the late steps of dense-core vesicle exocytosis. What remains unknown is the individual and collective impact of these proteins on the size of the releasable pools and the rate at which vesicles are primed into these pools. Chromaffin cells isolated from Rab3A-/-, Rab27-/-, and double knock-out mice were used to examine the density of docked vesicles, granule mobility at the membrane, and exocytotic activity. Docked granules were visualized using electron micrographs and quantified based on their placement with respect to the plasma membrane. Mobility of granules was examined using total internal reflection fluorescence (TIRF) microscopy with virus-infected chromaffin cells expressing NPY-cherry. Changes in membrane capacitance were recorded from chromaffin cells in adrenal slices using patch clamp electrophysiology, providing data reflecting fusion kinetics and the refilling of releasable pools. The present study has identified the contributions of Rab3 and Rab27 to release readiness of dense-core vesicles in primary mouse chromaffin cells.

Caveolin and Cholesterol Control of Mechanosensitive Channels in Muscle

In Duchene muscular dystrophy the loss of the cytoskeletal element dystrophin modifies the localization of sarcolemma components and the stress distribution to those components. There is a change in the distribution of caveolae and dysregulation of mechanosensitive ion channels (MSCs) both of which may contribute to the elevated Ca2+ levels present in dystrophic myotubes. Caveolae are curved cholesterol rich membrane structures associated with dystrophin that contain many signaling molecules. We have shown that overexpression of Caveolin-3 in mouse myotubes increases the incidence of MSCs in patches and average MSC current. However, it has no effect on their kinetics of activation. Cholesterol depletion by MβCD produces an even larger increase in MSC incidence and average patch current, and also significantly decreases the relaxation rate of the membrane as observed by membrane capacitance changes. We have used miRNA against TRP channels (TRPC1, TRPC4, TRPC6, TRPV2) that have been reported to mechanosensitive in various systems to try and determine the identity of the channel revealed by cholesterol depletion. Average patch current and immunofluorescence staining has been used to determine the level of knockdown of the individual TRP channel subunits.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Deletion of Fas in the pancreatic β-cells leads to enhanced insulin secretion

Fas/Fas ligand belongs to the tumor necrosis factor superfamily of receptors/ligands and is best known for its role in apoptosis. However, recent evidence supports its role in other cellular responses, including proliferation and survival. Although Fas has been implicated as an essential mediator of beta-cell death in the pathogenesis of type 1 diabetes, the essential role of Fas specifically in pancreatic beta-cells has been found to be controversial. Moreover, the role of Fas on beta-cell homeostasis and function is not clear. The objective of this study is to determine the role of Fas specifically in beta-cells under both physiological and diabetes models. Mice with Fas deletion specifically in the beta-cells were generated using the Cre-loxP system. Cre-mediated Fas deletion was under the control of the rat insulin promoter. Absence of Fas in beta-cells leads to complete protection against FasL-induced cell death. However, Fas is not essential in determining beta-cell mass or susceptibility to streptozotocin- or HFD-induced diabetes. Importantly, Fas deletion in beta-cells leads to increased p65 expression, enhanced glucose tolerance, and glucose-stimulated insulin secretion, with increased exocytosis as manifested by increased changes in membrane capacitance and increased expression of Syntaxin1A, VAMP2, and munc18a. Together, our study shows that Fas in the beta-cells indeed plays an essential role in the canonical death receptor-mediated apoptosis but is not essential in regulating beta-cell mass or diabetes development. However, beta-cell Fas is critical in the regulation of glucose homeostasis through regulation of the exocytosis machinery.

Two Disaccharides and Trimethylamine N-Oxide Affect Aβ Aggregation Differently, but All Attenuate Oligomer-Induced Membrane Permeability

Interaction between aggregates of amyloid beta protein (Abeta) and membranes has been hypothesized by many to be a key event in the mechanism of neurotoxicity associated with Alzheimer's disease (AD). Proposed membrane-related mechanisms of neurotoxicity include ion channel formation, membrane disruption, changes in membrane capacitance, and lipid membrane oxidation. Recently, osmolytes such as trehalose have been found to delay Abeta aggregation in vitro and reduce neurotoxicity. However, no direct measurements have separated the effects of osmolytes on Abeta aggregation versus membrane interactions. In this article, we tested the influence of trehalose, sucrose and trimethylamine-N-oxide (TMAO) on Abeta aggregation and fluorescent dye leakage induced by Abeta aggregates from liposomes. In the absence of lipid vesicles, trehalose and sucrose, but not TMAO, were found to delay Abeta aggregation. In contrast, all of the osmolytes significantly attenuated dye leakage. Dissolution of preformed Abeta aggregates was excluded as a possible mechanism of dye leakage attenuation by measurements of Congo red binding as well as hydrogen-deuterium exchange detected by mass spectrometry (HX-MS). However, the accelerated conversion of high order oligomers to fibril caused by vesicles did not take place if any of the three osmolytes presented. Instead, in the case of disaccharide, osmolytes were found to form adducts with Abeta, and change the dissociation dynamics of soluble oligomeric species. Both effects may have contributed to the observed osmolyte attenuation of dye leakage. These results suggest that disaccharides and TMAO may have very different effects on Abeta aggregation because of the different tendencies of the osmolytes to interact with the peptide backbone. However, the effects on Abeta membrane interaction may be due to much more general phenomena associated with osmolyte enhancement of Abeta oligomer stability and/or direct interaction of osmolyte with the membrane surface.

Cab45b, a Munc18b-interacting Partner, Regulates Exocytosis in Pancreatic β-Cells

Cab45b is a cytosolic Ca(2+)-binding protein reported to regulate zymogen secretion in pancreatic acini. We now show that Cab45b is also expressed in pancreatic islet beta-cells and interacts there with the Sec1-Munc18 protein Munc18b. We employed patch clamp cell capacitance measurements to show that antibodies against Cab45b inhibited depolarization-evoked membrane capacitance increments, suggesting an impact on beta-cell granule exocytosis, both the readily releasable granule pool and refilling of this pool. Site-specific mutants in the Cab45b EF-hands were used to dissect the molecular interactions involved in Cab45b function. Mutants in EF-hands 2 and 3 had no detectable effects on interaction of Cab45b with Munc18b and did not affect the depolarization-evoked calcium currents, but remarkably, they facilitated the complex formation of Munc18b with syntaxin-2 and -3. As a result, these two EF-hand mutants inhibited beta-cell membrane capacitance increments. This inhibition is mediated via Munc18b because Munc18b silencing with small interfering RNA abolished the effects of these two mutants. The results suggest a mechanism for Cab45b action that involves regulating the dynamic association of Munc18b with SNAREs to impact beta-cell granule exocytosis.

Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Dynasore inhibits rapid endocytosis in bovine chromaffin cells

Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 s to a value lower than the prestimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve fitted to the decay phase were all approximately 110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis.

Somatostatin release, electrical activity, membrane currents and exocytosis in human pancreatic delta cells

The aim of this study was to characterise electrical activity, ion channels, exocytosis and somatostatin release in human delta cells/pancreatic islets. Glucose-stimulated somatostatin release was measured from intact human islets. Membrane potential, currents and changes in membrane capacitance (reflecting exocytosis) were recorded from individual human delta cells identified by immunocytochemistry. Somatostatin secretion from human islets was stimulated by glucose and tolbutamide and inhibited by diazoxide. Human delta cells generated bursting or sporadic electrical activity, which was enhanced by tolbutamide but unaffected by glucose. Delta cells contained a tolbutamide-insensitive, Ba(2+)-sensitive inwardly rectifying K(+) current and two types of voltage-gated K(+) currents, sensitive to tetraethylammonium/stromatoxin (delayed rectifying, Kv2.1/2.2) and 4-aminopyridine (A current). Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents contributed to the action potential upstroke but TTX had no effect on somatostatin release. Delta cells are equipped with Ca(2+) channels blocked by isradipine (L), omega-agatoxin (P/Q) and NNC 55-0396 (T). Blockade of any of these channels interferes with delta cell electrical activity and abolishes glucose-stimulated somatostatin release. Capacitance measurements revealed a slow component of depolarisation-evoked exocytosis sensitive to omega-agatoxin. Action potential firing in delta cells is modulated by ATP-sensitive K(+)-channel activity. The membrane potential is stabilised by Ba(2+)-sensitive inwardly rectifying K(+) channels. Voltage-gated L- and T-type Ca(2+) channels are required for electrical activity, whereas Na(+) currents and P/Q-type Ca(2+) channels contribute to (but are not necessary for) the upstroke of the action potential. Action potential repolarisation is mediated by A-type and Kv2.1/2.2 K(+) channels. Exocytosis is tightly linked to Ca(2+)-influx via P/Q-type Ca(2+) channels. Glucose stimulation of somatostatin secretion involves both K(ATP) channel-dependent and -independent processes.