Interaction of microtubules and actin during the post-fusion phase of exocytosis.

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis.

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

Polymer-coated NaYF4:Yb³âº, Er³âº upconversion nanoparticles for charge-dependent cellular imaging.

Lanthanide-doped upconversion nanoparticles (UCNPs) are considered promising novel near-infrared (NIR) bioimaging agents with the characteristics of high contrast and high penetration depth. However, the interactions between charged UCNPs and mammalian cells have not been thoroughly studied, and the corresponding intracellular uptake pathways remain unclear. Herein, our research work involved the use of a hydrothermal method to synthesize polyvinylpyrrolidone-coated UCNPs (UCNP-PVP), and then a ligand exchange reaction was performed on UCNP-PVP, with the help of polyethylenimine (PEI) and poly(acrylic acid) (PAA), to generate UCNP-PEI and UCNP-PAA. These polymer-coated UCNPs demonstrated good dispersibility in aqueous medium, had the same elemental composition and crystal phase, shared similar TEM and dynamic light scattering (DLS) size distribution, and exhibited similar upconversion luminescence efficiency. However, the positively charged UCNP-PEI evinced greatly enhanced cellular uptake in comparison with its neutral or negative counterparts, as shown by multiphoton confocal microscopy and inductively coupled plasma mass spectrometry (ICP-MS) measurements. Meanwhile, we found that cationic UCNP-PEI can be effectively internalized mainly through the clathrin endocytic mechanism, as revealed by colocalization, chemical, and genetic inhibitor studies. This study elucidates the role of the surface polymer coatings in governing UCNP-cell interactions, and it is the first report on the endocytic mechanism of positively charged lanthanide-doped UCNPs. Furthermore, this study provides important guidance for the development of UCNPs as specific intracellular nanoprobes, allowing us to control the UCNP-cell interactions by tuning surface properties.

Early events induced by chitosan on plant cells.

Chitosan (a polymer of beta-1,4-glucosamine residues) is a deacetylated derivative of chitin which presents antifungal properties and acts as a potent elicitor of plant resistance against fungal pathogens. Attention was focused in this study on the chitosan-induced early events in the elicitation chain. Thus, it was shown that chitosan triggered in a dose-dependent manner rapid membrane transient depolarization of Mimosa pudica motor cells and, correlatively, a transient rise of pH in the incubation medium of pulvinar tissues. By using plasma membrane vesicles (PMVs), it was specified that a primary site of action of the compound is the plasma membrane H(+)-ATPase as shown by its inhibitory effect on the proton pumping and the catalytic activity of the enzyme up to 250 microg ml(-1). As a consequence, chitosan treatment modified H(+)-mediated processes, in particular it inhibited the uptake of the H(+)-substrate co-transported sucrose and valine, and inhibited the light-induced H(+)/K(+)-mediated turgor reaction of motor cells. The present data also allowed the limit of the cytotoxicity of the compound to be established close to a concentration of 100 microg ml(-1) at the plasma membrane level. As a consequence, chitosan could be preferably used in plant disease control as a powerful elicitor rather than a direct antifungal agent.

Trafficking the NGF signal: implications for normal and degenerating neurons.

Nerve growth factor (NGF) activates TrkA to trigger signaling events that promote the survival, differentiation and maintenance of neurons. The mechanism(s) that controls the retrograde transport of the NGF signal from axon terminals to neuron cell bodies is not known. The 'signaling endosome' hypothesis stipulates that NGF, TrkA and signaling proteins are retrogradely transported on endocytic vesicles. Here, we provide evidence for the existence of signaling endosomes. Following NGF treatment, clathrin-coated vesicles (CCVs) contain NGF bound to TrkA together with activated signaling proteins of the Ras/pErk1/2 pathway. NGF signals from isolated CCVs through the Erk1/2 pathway. Early endosomes appear to represent a second type of signaling endosomes. We found that NGF induced a sustained activation of Rap1, a small monomeric GTP-binding protein of the Ras family, and that this activation occurred in early endosomes that contain key elements of Rap1/pErk1/2 pathway. We discuss the possibility that the failure of retrograde NGF signaling in a mouse model of Down syndrome (Ts65Dn) may be due to the failure to retrograde transport signaling endosomes. It is important to define further the significance of signaling endosomes in the biology of both normal and degenerating neurons.

Acid-base effects on intestinal Na(+) absorption and vesicular trafficking.

We examined for vesicular trafficking of the Na(+)/H(+) exchanger (NHE) in pH-stimulated ileal and CO(2)-stimulated colonic Na(+) absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at x27,500 magnification. Internalization of ileal apical membranes labeled with FITC-phytohemagglutinin was assessed using confocal microscopy, and pH-stimulated ileal Na(+) absorption was measured after exposure to wortmannin. Apical membrane protein biotinylation of ileal and colonic segments and Western blots of recovered proteins were performed. In ileal epithelial cells incubated in HCO/Ringer or HEPES/Ringer solution, the number of subapical vesicles, the relative quantity of apical membrane NHE isoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membrane fluorescence under the confocal microscope were not affected by pH values between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulated ileal Na(+) absorption. In colonic epithelial apical membranes, NHE3 protein content was greater at a PCO(2) value of 70 than 21 mmHg, was internalized when PCO(2) was reduced, and was exocytosed when PCO(2) was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na(+) absorption but is important in CO(2)-stimulated colonic Na(+) absorption.

Exiting the endoplasmic reticulum.

The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments. Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes. Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane. Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments. Three classes of cytoplasmic coats have been identified. (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells [reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic. Ann. Rev. Cell. Dev. Biol. 11, 677--706.)]. (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER). It may also participate in anterograde transport from the ER [reviewed in (Aridor, M., Balch, W.E., 1999. Integration of endoplasmic reticulum signaling in health and disease. Nature 5, 745--751.)]. (3) COPII coats mediate anterograde transport of cargo out of the ER [Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895--907; Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membrane traffic. Int. Rev. Cytol. 195, 67--144.]. The COPII coat is required for budding from the ER and ER to Golgi trafficking. Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle. Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles.

Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Effect of lamivudine on uptake of organic cations by rat renal brush-border and basolateral membrane vesicles.

The effect of lamivudine on uptake of a representative organic cation, tetraethylammonium (TEA), by rat renal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) has been investigated. The pH-driven uptake of TEA by BBMV (pHin = 6.0, pHout = 7.5) was inhibited by lamivudine. The IC50 value (concentration resulting in 50% inhibition) for the concentration-dependent effect of lamivudine on TEA uptake by BBMV after 30 s was 2668 microM whereas IC50 values for cimetidine and trimethoprim were < 2.5 microM and < 25 microM, respectively. The early uptake of TEA by BLMV was also reduced significantly by lamivudine. The IC50 value for the concentration-dependent effect of lamivudine on uptake of TEA by BLMV at 30 s was > 25 mM, whereas the IC50 values for cimetidine and trimethoprim were 2116 microM and 445 microM, respectively. These findings suggest that compared with other cationic drugs, such as trimethoprim and cimetidine, lamivudine is a weak inhibitor of organic cation transport into the tubules by the brush-border and basolateral membranes of renal epithelial cells. It is unlikely lamivudine will have any significant effect on the excretion of co-administered cationic drugs by the renal tubules.

COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo.

Microinjection of the slowly hydrolyzable GTP analogue GTP(gamma)S or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15 degrees C and by microinjection of antibodies against (beta)-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTP(gamma)S or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTP(gamma)S and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.

ERG30, a VAP-33-related protein, functions in protein transport mediated by COPI vesicles.

Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein-endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)-which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH(2) terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.

Vesicle-associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking.

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

The 26-mer peptide released from SNAP-25 cleavage by botulinum neurotoxin E inhibits vesicle docking.

Botulinum neurotoxin E (BoNT E) cleaves SNAP-25 at the C-terminal domain releasing a 26-mer peptide. This peptide product may act as an excitation-secretion uncoupling peptide (ESUP) to inhibit vesicle fusion and thus contribute to the efficacy of BoNT E in disabling neurosecretion. We have addressed this question using a synthetic 26-mer peptide which mimics the amino acid sequence of the naturally released peptide, and is hereafter denoted as ESUP E. This synthetic peptide is a potent inhibitor of Ca2+-evoked exocytosis in permeabilized chromaffin cells and reduces neurotransmitter release from identified cholinergic synapses in in vitro buccal ganglia of Aplysia californica. In chromaffin cells, both ESUP E and BoNT E abrogate the slow component of secretion without affecting the fast, Ca2+-mediated fusion event. Analysis of immunoprecipitates of the synaptic ternary complex involving SNAP-25, VAMP and syntaxin demonstrates that ESUP E interferes with the assembly of the docking complex. Thus, the efficacy of BoNTs as inhibitors of neurosecretion may arise from the synergistic action of cleaving the substrate and releasing peptide products that disable the fusion process by blocking specific steps of the exocytotic cascade.

A molecular basis for retinol stimulation of vesicle budding in vivo and in vitro.

Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane thiols. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.

A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex.

Reconstitution of synaptic vesicle formation in vitro has revealed a pathway of synaptic vesicle biogenesis from endosomes that requires the heterotetrameric adaptor complex AP3. Because synaptic vesicles have a distinct protein composition, the AP3 complex should selectively recognize some or all of the synaptic vesicle proteins. Here we show that one element of this recognition process is the v-SNARE, VAMP-2, because tetanus toxin, which cleaves VAMP-2, inhibited the formation of synaptic vesicles and their coating with AP3 in vitro. Mutant tetanus toxin and botulinum toxins, which cleave t-SNAREs, did not inhibit synaptic vesicle production. AP3-containing complexes isolated from coated vesicles could be immunoprecipitated by a VAMP-2 antibody. These data imply that AP3 recognizes a component of the fusion machinery, which may prevent the production of inert synaptic vesicles.

Alzheimer's amyloid-beta peptide inhibits sodium/calcium exchange measured in rat and human brain plasma membrane vesicles.

Na+/Ca2+ exchange activity was measured by monitoring vesicular Ca2+ content after incubation in buffers containing 45Ca2+. When Na+-loaded vesicles were placed into Na+-free buffer, vesicular Ca2+ content increased rapidly and reached a plateau after two to three minutes. Only preaggregated amyloid-beta1-40 (Abeta1-40) and Abeta25-35 reduced vesicular Ca2+ content. Both peptides produced a maximal reduction in Ca2+ content of approximately 50%. The peptides reduced Ca2+ content with similar potency and half maximal effects were seen at less than 10 microM for Abeta25-35. Calcium-loaded vesicles mediate a rapid Ca2+/Ca2+ exchange, which also was inhibited by aggregated Abeta25-35. Aggregated Abeta25-35 did not affect the passive Ca2+ permeability of the vesicles. Aggregated Abeta25-35 reduced Ca2+ content in plasma membrane vesicles isolated from normal and Alzheimer's disease frontal cortex with less potency but the same efficacy as seen in rat brain. Aggregated Abeta25-35 did not produce nonspecific effects on vesicle morphology such as clumping or loss of intact vesicles. When placed in the buffer used to measure Ca2+ content, Congo Red at molar ratios of less than one blocked the inhibitory effect of preaggregated Abeta25-35. When added in equimolar concentrations to freshly dissolved and unaggregated Abeta25-35, Congo Red also was effective at blocking the inhibitory effect on Ca2+ content. In contrast, vitamin E (antioxidant) and N-tert-butyl-alpha-phenylnitrone (spin trapping agent) failed to block the inhibitory action of aggregated Abeta25-35. The exact mechanisms of Abeta-induced neurotoxicity in cell culture has yet to be solved. Accumulation of free radicals play a necessary role, but disruptions of Ca2+ homeostasis are also important. The data presented here are consistent with a proposed mechanism where aggregated Abeta peptides directly interact with hydrophobic surfaces of the exchanger protein and/or lipid bilayer and interfere with plasma membrane Ca2+ transport.

Effect of urea on biomimetic aggregates.

The effect of urea on biomimetic aggregates (aqueous and reversed micelles, vesicles and monolayers) was investigated to obtain insights into the effect of the denaturant on structured macromolecules. Direct evidence obtained from light scattering (static and dynamic), monolayer maximum isothermal compression and ionic conductivity measurements, together with indirect evidence from fluorescence photodissociation, fluorescence suppression, and thermal reactions, strongly indicates the direct interaction mechanism of urea with the aggregates. Preferential solvation of the surfactant headgroups by urea results in an increase in the monomer dissociation degree (when applied), which leads to an increase in the area per headgroup and also in the loss of counterion affinities.

Desensitization and internalization of adenosine A1 receptors in rat brain by in vivo treatment with R-PIA: involvement of coated vesicles.

Chronic treatment of rats with R-PIA 'in vivo' desensitized adenosine A1 receptor-mediated inhibition of adenylyl cyclase in brain plasma membranes and increased basal and forskolin-stimulated adenylyl cyclase. The adenosine A1 receptor agonist CHA (cyclohexyl adenosine) inhibited forskolin-stimulated adenylyl cyclase in synaptic plasma membranes from control rats but failed to do so in membranes isolated from rats treated with R-PIA. This loss of response was accompanied with a significant decrease in both, total number of adenosine A1 receptors and steady-state level of alpha-Gi in synaptic plasma membranes. An increase in the steady-state level of alpha-Gs in synaptic plasma membranes was also observed by R-PIA treatment. Concurrently, a significant increase of adenosine A1 receptors was observed in microsomes and coated vesicles. These results demonstrate adenosine A1 receptor desensitization in rat brain by 'in vivo' treatment with R-PIA and suggest a role for coated vesicles in the internalization of G-protein coupled receptors.

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: modulation by calcium and GTP gamma S.

This study reports the analysis of K+ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K+ channels were observed with amplitudes of 25-30, 45-55, and 80-100 pS, all of which exhibited mean open-times of 1-2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K+ channel activity was regulated by Ca2+. Little or no change in activity was observed when Ca2+ was added to the cis side of the bilayer. Addition of 10 microM total Ca2+ also resulted in little change in K+ channel activity. However, at 80 microM total Ca2+ all K+ channel activity was suppressed along with the activation of a 100 pS Cl- channel. The K+ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTP gamma S to the trans side of the bilayer resulted in a restriction of activity to the 45-50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K+ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the alpha subunits of G0, Gs alpha, and Gi alpha and the low molecular weight G protein, ras.(ABSTRACT TRUNCATED AT 250 WORDS)