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The adrenal medulla plays a critical role in mammalian homeostasis and the stress response. It is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with peptides into the bloodstream affecting distant target organs. Despite been heavily studied, the central control of adrenal medulla and in-situ spatiotemporal responsiveness remains poorly understood. For this work, we continuously monitored the electrical activity of individual adrenomedullary chromaffin cells in the living anesthetized rat using multielectrode arrays. We measured the chromaffin cell activity under basal and physiological stress conditions and characterized the functional micro-architecture of the adrenal medulla. Under basal conditions, chromaffin cells fired action potentials with frequencies between ~0.2 and 4 Hz. Activity was almost completely driven by sympathetic inputs coming through the splanchnic nerve. Chromaffin cells were organized into independent local networks in which cells fired in a specific order, with latencies from hundreds of microseconds to a few milliseconds. Electrical stimulation of the splanchnic nerve evoked almost exactly the same spatiotemporal firing patterns that occurred spontaneously. Hypoglycemic stress, induced by insulin administration resulted in increased activity of a subset of the chromaffin cells. In contrast, respiratory arrest induced by lethal anesthesia resulted in an increase in the activity of virtually all chromaffin cells before cessation of all activity. These results suggest a stressor-specific activation of adrenomedullary chromaffin cell networks and revealed a surprisingly complex electrical organization that likely reflects the dynamic nature of the adrenal medulla's neuroendocrine output during basal conditions and during different types of physiological stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lopez Ruiz, Ernst, Holz and Stuenkel.)

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10 -10 cm 2 /s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.

Central to the exocytotic release of hormones and neurotransmitters is the interaction of four SNARE motifs in proteins on the secretory granule/synaptic vesicle membrane (synaptobrevin/VAMP, v-SNARE) and on the plasma membrane (syntaxin and SNAP25, t-SNAREs). The interaction is thought to bring the opposing membranes together to enable fusion. An underlying motivation for this Viewpoint is to synthesize from recent diverse studies possible new insights about these events. We focus on a recent paper that demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion. Remarkably, lipid-perturbing agents rescue a palmitoylation-deficient mutant whose phenotype includes slow fusion pore expansion, suggesting that protein-protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein-lipid interactions leading to the fusion reaction. Unexpectedly, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE "acceptor" complex for synaptobrevin2. This insight, together with biophysical and optical studies from other laboratories, suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the subsequent trans-SNARE complex with the v-SNARE synaptobrevin form within 100 ms before fusion., (© 2020 Holz and Bittner.)

Fusion of secretory granules and synaptic vesicles with the plasma membrane is driven by SNARE protein interactions. Intensive investigations in vitro, which include x-ray crystallography, cryoelectron microscopy, and NMR analyses by numerous groups, have elucidated structures relevant to the function of these proteins. Although function depends on the proteins being membrane bound, for experimental reasons, most of the studies have used cytosolic domains, as exemplified by the groundbreaking studies that elucidated the structure of a tetrapeptide helical bundle formed by interaction of the cytosolic domains of syntaxin1A, SNAP25 (two peptides) and synaptobrevin 2. Because the cytosolic fragments were unfettered by membrane attachments, it is likely that the tetrapeptide helical bundle reflects the lowest energy state, such as that found in the "cis" interactions of the SNARE motifs after fusion when they co-localize in the plasma membrane. Much more difficult to study and still poorly understood are critical "trans" interactions between the synaptic vesicle SNARE protein synaptobrevin 2 and the plasma membrane syntaxin1A/SNAP25 complex that initiate the fusion event. In a series of articles from the laboratory of Lukas Tamm, the spontaneous orientation of the SNARE motif of membrane-bound, full-length syntaxin1A with respect to the membrane hosting syntaxin's transmembrane domain was investigated with nanometer precision under a variety of conditions, including those that model aspects of the "trans" configuration. The studies rely on fluorescence interference-contrast microscopy, a technique that utilizes the pattern of constructive and destructive interference arising from incoming and reflected excitation and emission light at the surface of a silicon chip that has been layered with oxidized silicon of varying depths. This Perspective discusses their findings, including the unexpected influence of the degree of lipid unsaturation on the orientation of the SNARE complex., (Copyright © 2019 Biophysical Society. All rights reserved.)

α-Synuclein is strongly implicated in the pathogenesis of Parkinson's disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of α-, β-, and γ-synuclein contains six to seven 11-amino acid repeats that are predicted to form amphipathic helices. Membrane-binding and membrane-curving abilities of synuclein raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous β-synuclein associates with secretory granules. Endogenous α-synuclein only rarely co-localizes with secretory granules. Overexpression of α-synuclein but not β-synuclein quickened the post- fusion discharge of BDNF-pHluorin by approxinately 30%. However, neither α- nor β-synuclein significantly altered curvature dynamics associated with fusion pore expansion that were measured by the combination of polarization and total internal reflection fluorescence microscopy (pTIRFM). Whatever the mechanism, the physiological significance of the small increased rate of post-fusion protein discharge caused by α-synuclein remains to be demonstrated, especially since endogenous β-, but not α-synuclein is the predominant synuclein isoform associated with chromaffin granules., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP-containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion., (© 2019 Abbineni et al.)

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species., (© 2017 Bohannon et al.)

The refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only its extracellular action but also the characteristics of its presentation to the extracellular milieu., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles "classical" receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (∼300-nm diameter) and typical clathrin-coated vesicles (∼90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments.

Assays for real-time investigation of exocytosis typically measure what is released from the granule. From this, inferences are made about the dynamics of membrane remodeling as fusion progresses from start to finish. We have recently undertaken a different approach to investigate the fusion process, by focusing not primarily on the granule, but rather its partner in exocytosis - the plasma membrane. We have been guided by the idea that biochemical interactions between the granule and plasma membranes before and during fusion, cause changes in membrane conformation. To enable study of membrane conformation, a novel imaging technique was developed combining polarized excitation of an oriented membrane probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI) with total internal reflection fluorescence microscopy (pTIRFM). Because this technique measures changes in membrane conformation (or deformations) directly, its usefulness persists even after granule cargo reporter (catecholamine, or protein), is no longer present. In this mini-review, we first summarize the workings of pTIRFM. We then discuss the application of the technique to investigate deformations in the membrane preceding fusion, and later, during fusion pore expansion. Finally, we discuss how expansion of the fusion pore may be regulated by the GTPase activity of dynamin., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Voltage-gated potassium (Kv) channels are critical for neuronal excitability and are targeted to specific subcellular compartments to carry out their unique functions. While it is widely believed that Kv channels exist as heteromeric complexes in neurons, direct tests of the hypothesis that specific heteromeric channel populations display divergent spatial and temporal dynamics are limited. Using a bimolecular fluorescence complementation approach, we monitored the assembly and localization of cell surface channel complexes in living cells. While PSD95-mediated clustering was subunit independent, selective visualization of heteromeric Kv complexes in rat hippocampal neurons revealed subunit-dependent localization that was not predicted by analyzing individual subunits. Assembly of Kv1.1 with Kv1.4 prevented axonal localization but not surface expression, while inclusion of Kv1.2 imparted clustering at presynaptic sites and decreased channel mobility within the axon. This mechanism by which specific Kv channel subunits can act in a dominant manner to impose unique trafficking properties to heteromeric complexes extended to Shab-related family of Kv channels. When coexpressed, Kv2.1 and Kv2.2 heteromultimers did not aggregate in somatodendritic clusters observed with expression of Kv2.1 alone. These studies demonstrate selective axonal trafficking and surface localization of distinct Kv channels based on their subunit composition.

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.

We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane intermediate is regulated by divalent cation. When Sr2+ is used instead of Ca2+ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation.

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane-cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane-plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

It has previously been shown that misfolded mutant Akita proinsulin in the endoplasmic reticulum engages directly in protein complexes either with nonmutant proinsulin or with "hProCpepGFP" (human proinsulin bearing emerald-GFP within the C-peptide), impairing the trafficking of these "bystander" proinsulin molecules (Liu, M., Hodish, I., Rhodes, C. J., and Arvan, P. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 15841-15846). Herein, we generated transgenic mice, which, in addition to expressing endogenous proinsulin, exhibit beta-cell-specific expression of hProCpepGFP via the Ins1 promoter. In these mice, hProCpepGFP protein levels are physiologically regulated, and hProCpepGFP is packaged and processed to CpepGFP that is co-stored in beta-secretory granules. Visualization of CpepGFP fluorescence provides a quantifiable measure of pancreatic islet insulin content that can be followed in live animals in states of health and disease. We examined loss of pancreatic insulin in hProCpepGFP transgenic mice mated to Akita mice that develop neonatal diabetes because of the expression of misfolded proinsulin. Loss of bystander insulin in Akita animals is detected initially as a block in CpepGFP/insulin production with intracellular accumulation of the precursor, followed ultimately by loss of pancreatic beta-cells. The data support that misfolded proinsulin perturbs bystander proinsulin in the endoplasmic reticulum, leading to beta-cell failure.

Rapid treatment (1 min) of rat striatal synaptosomes with low-dose amphetamine increases surface expression of the dopamine transporter (DAT). Using mouse neuroblastoma N2A cells, stably transfected with green fluorescent protein-DAT, we demonstrate the real-time substrate-induced rapid trafficking of DAT to the plasma membrane using total internal reflection fluorescence microscopy (TIRFM). Both the physiological substrate, dopamine, and amphetamine began to increase surface DAT within 10 s of drug addition and steadily increased surface DAT until removal 2 min later. The substrate-induced rise in surface DAT was dose-dependent, was blocked by cocaine, and abated after drug removal. Although individual vesicle fusion was not visually detectable, exocytosis of DAT was blocked using both tetanus neurotoxin and botulinum neurotoxin C to cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Notably, the dopamine-induced increase in surface DAT was cocaine-sensitive but D(2)-receptor independent. TIRFM data were confirmed in human DAT-N2A cells using biotinylation, and similar effects were detected in rat striatal synaptosomes. A specific inhibitor of protein kinase C-beta blocked the substrate-mediated increase in surface DAT in both DAT-N2A cells and rat striatal synaptosomes. These data demonstrate that the physiological substrate, dopamine, and amphetamine rapidly increase the trafficking of DAT to the surface by a mechanism dependent on SNARE proteins and protein kinase C-beta but independent of dopamine D(2) receptor activation. Importantly, this study suggests that the reuptake system is poised to rapidly increase its function during dopamine secretion to tightly regulate dopaminergic neurotransmission.

We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDS-resistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

Our current notions of different granule pools, granule interaction with the plasma membrane, and ultimately granule and plasma membrane soluble N-ethylmaleimide-sensitive-factor attachment protein (SNARE) interactions, result largely from inferences based upon biochemical alterations of secretion kinetics. Another view of events comes from studies using total internal reflection fluorescence microscopy (TIRFM) to investigate granule behaviour immediately adjacent to the plasma membrane. The motions of secretory (chromaffin) granules in bovine chromaffin cells visualized by TIRFM are highly restricted, as if granules are caged or tethered. These small motions are regulated by ATP and Ca2+, two factors that increase priming of the secretory response. There is no evidence that granules decrease their motion immediately before secretion. To the contrary, there is a tendency for granules to increase their motions and travel within a few hundred milliseconds of fusion. Hence, the notion of a long-lived docked state as a prelude to fusion does not encompass the physical reality of molecular scale motions, multiple tethering states and significant travel immediately preceding the exocytotic event. Increased travel may increase the probability of granules interacting productively with the plasma membrane constituents, thereby, increasing the probability of fusion.

The state of secretory granules immediately before fusion with the plasma membrane is unknown, although the granules are generally assumed to be stably bound (docked). We had previously developed methods using total internal reflection fluorescence microscopy and image analysis to determine the position of chromaffin granules immediately adjacent to the plasma membrane with high precision, often to within approximately 10 nm, or <5% of the granule diameter (300 nm). These distances are of the dimensions of large proteins and are comparable with the unitary step sizes of molecular motors. Here we demonstrate with quantitative measures of granule travel in the plane parallel to the plasma membrane that secretory granules change position within several hundred milliseconds of nicotinic agonist-induced fusion. Furthermore, just before fusion, granules frequently move to areas that they have rarely visited. The movement of granules to new areas is most evident for granules that fuse later during the stimulus. The movement may increase the probability of productive interactions of the granule with the plasma membrane or may reflect the pull of molecular interactions between the granule and the plasma membrane that are part of the fusion process. Thus, instead of being stably docked before exocytosis, granules undergo molecular-scale motions and travel immediately preceding the fusion event.

The contribution by Micheva et al. in this issue of Molecular Pharmacology adds to our understanding of the action of pregabalin, a drug used for treatment of partial seizures and neuropathic pain. The authors examine the effects of pregabalin on presynaptic function of cultured hippocampal neurons using a powerful technique to follow the trafficking of synaptic vesicles in individual boutons. The study revealed that pregabalin reduces the readily releasable pool of synaptic vesicles in an N-methyl-d-aspartate receptor-dependent manner.

1. Time with Julie in his laboratory at the NIH in the early 1970s is remembered. The experience led to a life-long interest in the regulation of catecholamine secretion. Here are summarized aspects of this work. 2. The relationship between ATP-dependent priming of exocytosis and the polyphosphoinositides is reviewed. In addition, studies are summarized in which total internal reflection fluorescent microscopy (TIRFM) was used to visualize secretory granule behavior before exocytosis and individual exocytotic events. 3. Quantitative optical analysis indicates that chromaffin granule motion is highly restricted but regulated. Granules can undergo significant motion in the 100 ms prior to fusion and interactions with the plasma membrane leading to fusion can occur within this time. The small motions may permit granules adjacent to the plasma membrane to repetitively sample microdomains of the plasma membrane, thereby increasing the probability of fruitful interactions that lead to fusion.

Total internal reflection fluorescence microscopy was used to monitor changes in individual granule motions related to the secretory response in chromaffin cells. Because the motions of granules are very small (tens of nanometers), instrumental noise in the quantitation of granule motion was taken into account. ATP and Ca2+, both of which prime secretion before fusion, also affect granule motion. Removal of ATP in permeabilized cells causes average granule motion to decrease. Nicotinic stimulation causes a calcium-dependent increase in average granule motion. This effect is more pronounced for granules that undergo exocytosis than for those that do not. Fusion is not preceded by a reduction in mobility. Granules sometimes move 100 nm or more up to and within a tenth of a second before fusion. Thus, the jittering motion of granules adjacent to the plasma membrane is regulated by factors that regulate secretion and may play a role in secretion. Motion continues until shortly before fusion, suggesting that interaction of granule and plasma membrane proteins is transient. Disruption of actin dynamics did not significantly alter granule motion.

It has long been believed that the cortical actin cytoskeleton plays an important role in regulating the secretion of hormones and neurotransmitters. In this study, we investigated the control of actin dynamics in primary neuroendocrine cells and determined the relationship of actin dynamics to various components of the secretory response. The amount of cortical f-actin in chromaffin cells was quantified in confocal images of cells stained with Alexa Fluor 568 phalloidin. Manipulations that decreased levels of phosphatidylinositol-4,5-bisphosphate (PIP(2)) (e.g., removal of ATP, the expression of a protein that can sequester PIP(2)) rapidly reduced the amount of cortical actin. In contrast, cytoskeletal disruptors such as latrunculin were much less able to reduce cortical actin levels, indicating that the amount of cortical f-actin depends more strongly on PIP(2) than on the availability of g-actin. Not only does PIP(2) regulate actin, but actin regulates the level of PIP(2), as revealed by PIP(2) labeling studies. Manipulation of cortical actin had differing effects on the ATP-dependent and -independent components of secretion. ATP-dependent secretion was particularly sensitive to changes in cortical actin stability and was inhibited by expression of a protein (Yersinia pestis protein kinase A) that disassembles cortical f-actin and by pharmacological agents that promote either disassembly or stabilization of actin. The data suggest that an ATP-dependent component of secretion requires rapid changes in actin dynamics. These results point to a complex web of interactions involving PIP(2), actin, and the secretory response.

Secretory granules labeled with Vamp-green fluorescent protein (GFP) showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy. In approximately 90% of fusion events, we observed a large increase in fluorescence intensity coupled with a transition from a small punctate appearance to a larger, spreading cloud with free diffusion of the Vamp-GFP into the plasma membrane. Quantitation suggests that these events reflect the progression of an initially fused and spherical granule flattening into the plane of the plasma membrane as the Vamp-GFP simultaneously diffuses through the fusion junction. Approximately 10% of the events showed a transition from puncta to ring-like structures coupled with little or no spreading. The ring-like images correspond quantitatively to granules fusing and retaining concavity (recess of approximately 200 nm). A majority of fusion events involved granules that were present in the evanescent field for at least 12 s. However, approximately 20% of the events involved granules that were present in the evanescent field for no more than 0.3 s, indicating that the interaction of the granule with the plasma membrane that leads to exocytosis can occur within that time. In addition, approximately 10% of the exocytotic sites were much more likely to occur within a granule diameter of a previous event than can be accounted for by chance, suggestive of sequential (piggy-back) exocytosis that has been observed in other cells. Overall granule behavior before and during fusion is strikingly similar to exocytosis previously described in the constitutive secretory pathway.

Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the zinc finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding. Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.

Sustained release of neurotransmitter depends upon the recycling of synaptic vesicles. Until now, it has been assumed that vesicle recycling is regulated by signals from the presynaptic bouton alone, but results from rat hippocampal neurons reported here indicate that this need not be the case. Fluorescence imaging and pharmacological analysis show that a nitric oxide (NO) signal generated postsynaptically can regulate endocytosis and at least one later step in synaptic vesicle recycling. The proposed retrograde pathway involves an NMDA receptor (NMDAR)-dependent postsynaptic production of NO, diffusion of NO to a presynaptic site, and a cGMP-dependent increase in presynaptic phosphatidylinositol 4,5-biphosphate (PIP2). These results indicate that the regulation of synaptic vesicle recycling may integrate a much broader range of neural activity signals than previously recognized, including postsynaptic depolarization and the activation of NMDARs at both immediate and nearby postsynaptic active zones.

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1-V12, Rac1-L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin-permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1-N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21-activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1-V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1-V12-mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin-A inhibited secretion and eliminated the secretory effects of Rac1-V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca2+ influx, partly through regulation of cytoskeletal organization.

Rim1 has been identified in brain by its ability to bind Rab3a-GTP and has been postulated to be a Rab3a effector protein. Like Rabphilin3, another putative Rab3a effector protein, Rim1 modulates secretion and contains a zinc-finger and two C2 domains. We have investigated the structural basis for the ability of Rim1 to bind Rab3a-GTP and to stimulate exocytosis in chromaffin cells.

A slow ATP-dependent priming step precedes a rapid, Ca(2+)-dependent triggering step in exocytosis in chromaffin cells and in most, if not all, differentiated secretory cells. A major component of ATP-dependent secretion in permeabilized cells reflects the maintenance of the polyphosphoinositides, especially PtdIns-4,5-P2. Here we summarize recent experiments with PH-GFP (binds to PtdIns-4,5-P2) that indicate that PtdIns-4,5-P2 is localized primarily on the plasma membrane in chromaffin cells, and that it is this pool that plays a role in exocytosis. It is demonstrated that transiently expressed PH-GFP inhibits secretion in subsequently permeabilized cells. Recent studies using total internal reflection fluorescent microscopy (TIRFM) to measure chromaffin granule motion adjacent to the plasma membrane are also summarized. The quantitative analysis indicates that chromaffin granule motion is highly restricted and suggests that chromaffin granules are caged or tethered immediately adjacent to the plasma membrane.

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis of secrctory vesicles or granules. Among the cell models that have provided insight into the molecular machinery underlying the successive steps of exocytosis, adrenal chromaffin cells have taken a prominent place. Thus, most of the molecular players that orchestrate the formation, targeting, docking, and fusion of secrctory granules have been identified in chromaffin cells. By offering the opportunity to combine the use of recent biophysical techniques allowing single-vesicle resolution and specific biochemical modifications in the protein machinery involved in exocytosis, chromaffn cells remain a powerful model to address new and still open questions in the field of secretion.

Receptor-like protein-tyrosine phosphatase sigma (PTPvarsigma) is essential for neuronal development and function. Here we report that PTPvarsigma is a target of alpha-latrotoxin, a strong stimulator of neuronal exocytosis. alpha-Latrotoxin binds to the cell adhesion-like extracellular region of PTPvarsigma. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPvarsigma ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPvarsigma are not required for the alpha-latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPvarsigma resemble two other previously described alpha-latrotoxin receptors, neurexin and CIRL. Thus, alpha-latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPvarsigma, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction.

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-delta1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.

Rim1 was identified in brain by its ability to bind Rab3a-GTP and has been postulated to be a Rab3a effector protein. Like Rabphilin3, it modulates secretion and contains a zinc finger and two C2 domains. We have investigated the structural basis for the ability of Rim1 to bind Rab3a-GTP and to stimulate exocytosis in chromaffin cells. Both full-length and N-terminal Rim1 enhance secretion 40-50% in both intact and permeabilized cells. The abilities of Rim1 to enhance secretion and to bind Rab3a-GTP reside on distinct and relatively small domains that act independently. A approximately 30-amino acid sequence immediately N-terminal of the zinc finger constitutes the minimal Rab3a-GTP binding domain. This short sequence is not found in Rabphilin3 and is entirely different from the zinc finger and flanking regions of Rabphilin3 that bind Rab3a-GTP. The zinc finger domain in Rim1 is unnecessary for Rab3a-GTP binding but, alone, enhances secretion. An analysis of the characteristics of the enhancement of secretion in permeabilized chromaffin cells indicates that N-terminal Rim1 does not alter the sensitivity of secretion to Ca(2+) but, instead, increases the rate of ATP-dependent priming of secretion.

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

alpha-Latrotoxin, a component of black widow spider venom, stimulates transmitter release from nerve terminals and intact chromaffin cells and enhances secretion from permeabilized chromaffin cells already maximally stimulated by Ca(2+). In this study we demonstrate that chromaffin cells contain a protein antigenically similar to the cloned Ca(2+)-independent receptor for alpha-latrotoxin. Although this receptor has homology to the secretin family of G-protein-linked receptors, pertussis toxin has no effect on the ability of alpha-latrotoxin to enhance secretion, suggesting that neither G(i) nor G(o) is involved in the response. Furthermore, in the absence of Ca(2+), alpha-latrotoxin does not stimulate polyphosphoinositide-specific phospholipase C. alpha-Latrotoxin specifically enhances ATP-dependent secretion in permeabilized cells. An in situ assay for protein kinase C reveals that alpha-latrotoxin augments the activation of protein kinase C by Ca(2+), and use of protein kinase inhibitors demonstrates that this activation is important for the toxin's enhancing effect. This enhancement of secretion requires Ca(2+) concentrations above 3 microm and is not supported by Ba(2+) or nonhydrolyzable guanine nucleotides, which do not stimulate protein kinase C. We conclude that alpha-latrotoxin stimulates secretion in permeabilized cells by regulating a Ca(2+)- and ATP-dependent event involving protein kinase C.

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.

alpha-Latrotoxin binding to the calcium-independent receptor for alpha-latrotoxin (CIRL-1), a putative G-protein-coupled receptor, stimulates secretion from chromaffin and PC12 cells. Using patch clamp techniques and microspectrofluorimetry, we demonstrate that the interaction of alpha-latrotoxin with CIRL-1 produces a high conductance channel that permits increases in cytosolic Ca(2+). alpha-Latrotoxin interaction with CIRL-1 transiently expressed in bovine chromaffin cells produced a 400-pS channel, which rarely closed under Ca(2+)-free conditions. The major effect of overexpressing CIRL-1 was to greatly increase the sensitivity of chromaffin cells to channel formation by alpha-latrotoxin. alpha-Latrotoxin interaction with CIRL-1 transiently overexpressed in non-neuronal human embryonic kidney 293 (HEK293) cells produced channels that were nearly identical with those observed in chromaffin cells. Channel currents were reduced by millimolar Ca(2+). At alpha-latrotoxin concentrations below 500 pM, channel formation occurred many seconds after binding of toxin to CIRL-1 indicating distinct steps in channel formation. In all cases there was a rapid, sequential addition of channels once the first channel appeared. An analysis of CIRL-1 mutants indicated that channel formation in HEK293 cells is unlikely to be transduced by a G-protein-dependent mechanism. alpha-Latrotoxin interaction with a fusion construct composed of the extracellular domain of CIRL-1 anchored to the membrane by the transmembrane domain of vesicular stomatitis virus glycoprotein, and with neurexin 1alpha, an alpha-latrotoxin receptor structurally unrelated to CIRL-1, produced channels virtually identical with those observed with wild-type CIRL-1. We propose that alpha-latrotoxin receptors recruit toxin to facilitate its insertion across the membrane and that alpha-latrotoxin itself controls the conductance properties of the channels it produces.

The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells. The Rab3 family consisting of four highly homologous isoforms is associated with secretory granules and synaptic vesicles. Many different types of experiments indicate that Rab3a is a negative regulator of exocytosis and that its GTP-bound form interacts with Rabphilin3, a possible effector. Overexpression of Rabphilin3 in chromaffin cells enhances secretion. We have investigated the expression, localization, and effects on secretion of the various members of the Rab3 family in bovine chromaffin and PC12 cells. We found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying degrees in PC12 cells and in a fraction enriched in chromaffin granule membranes from the adrenal medulla. Immunocytochemistry revealed that all members of the family when overexpressed in PC12 cells localize to secretory granules. Binding constants for the interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d with Rabphilin3 were comparable (Kd = 10-20 nM). Overexpression of each of the four members of the Rab3 family inhibited secretion. Mutations in Rab3a were identified that strongly impaired the ability of the GTP-bound form to interact with Rabphilin3. The mutated proteins inhibited secretion similarly to wild type Rab3a. Although Rab3a and Rabphilin3 are located on the same secretory granule or secretory vesicle and interact both in vitro and in situ, it is concluded that the inhibition of secretion by overexpression of Rab3a is unrelated to its ability to interact with Rabphilin3.

Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.

Stimulation of neurotransmitter release by alpha-latrotoxin requires its binding to the calcium-independent receptor of alpha-latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor. CIRL consists of two noncovalently bound subunits, p85, a heptahelical integral membrane protein, and p120, a large extracellular polypeptide with domains homologous to lectin, olfactomedin, mucin, the secretin receptor family, and a novel structural motif common for large orphan G protein-coupled receptors. The analysis of CIRL deletion mutants indicates that the high affinity alpha-latrotoxin-binding site is located within residues 467-891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120. The N-terminal lectin, olfactomedin, and mucin domains of p120 are not required for the interaction with alpha-latrotoxin. Soluble p120 and all its fragments, which include the 467-770 residues, bind alpha-latrotoxin with low affinity suggesting the importance of membrane-embedded p85 for the stabilization of the complex of the toxin with p120. Two COOH-terminal deletion mutants of CIRL, one with the truncated cytoplasmic domain and the other with only one transmembrane segment left of seven, supported both alpha-latrotoxin-induced calcium uptake in HEK293 cells and alpha-latrotoxin-stimulated secretion when expressed in chromaffin cells, although with a different dose dependence than wild-type CIRL and its N-terminal deletion mutant. Thus the signaling domains of CIRL are not critically important for the stimulation of exocytosis in intact chromaffin cells by alpha-latrotoxin.

In the present study we investigated the lipid binding characteristics of the C2 domains of Rabphilin3a. We found that the tandem C2 domain of Rabphilin3a specifically bound lipid vesicles containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in a Ca2+-dependent manner. There was little binding to vesicles containing PtdIns(3,4)P2 in the presence or absence of Ca2+. Binding to phosphatidylinositol 3,4,5-triphosphate-containing vesicles was similar to binding to PtdIns(4,5)P2-containing vesicles. The presence of physiological amounts of phosphatidylserine (PS) greatly potentiated the ability of PtdIns(4,5)P2 to cause vesicle binding. As with the C2 domains together, the binding of individual C2 domain of Rabphilin3a was much greater to PtdIns(4,5)P2-containing vesicles than PtdIns(3,4)P2-containing vesicles. Both C2 domains also bound 29 mol % PS-containing vesicles in a Ca2+-dependent manner. Because of the importance of the C2B domain in the enhancement of secretion from chromaffin cells by Rabphilin3a, its biochemistry was further investigated. The mutation of aspartates 657 and 659 to asparagines in C2B decreased Ca2+-dependent and increased Ca2+-independent vesicle binding, indicating the Ca2+ dependence of the domain is provided by aspartic acid residues in the putative Ca2+-binding pocket. A peptide from the COOH-terminal region of the C2B domain specifically inhibited ATP-dependent secretion from permeabilized chromaffin cells and the binding of Rabphilin3a to phosphatidylcholine/PS/PtdIns(4,5)P2-containing lipid vesicles, suggesting a role of this sequence in secretion through its ability to interact with acidic lipid vesicles.