Biomedical subjects
Publications and source records attributed to W Almers.
The past year has witnessed progress in identifying late steps in exocytosis that are so short-lived as to be difficult to study biochemically. Recent studies have also revealed a novel and surprisingly fast mechanism of endocytosis that may be triggered by a rise in the intracellular concentration of Ca2+ and that retrieves exocytosed membrane in seconds.
Ca(2+)-triggered exocytosis was studied in single rat melanotrophs and bovine chromaffin cells by capacitance measurements. Sustained exocytosis required MgATP, but even in the absence of MgATP, Ca2+ could trigger exocytosis of 2700 granules in a typical melanotroph and of 840 granules in a chromaffin cell. Granules undergoing ATP-independent exocytosis were similar in number to those appearing docked to the plasmalemma in quickly frozen unfixed sections (3300 in a melanotroph and 830 in a chromaffin cell). Most exocytosis required tens of seconds, but a small pool of granules was released in tens of milliseconds. Evidently, only a small subset of docked granules is rapidly releasable. We suggest that, temporally, the last ATP-dependent step in exocytosis is closely associated with docking and that docked granules reach fusion competence only after subsequent steps.
Several proteins involved in exocytosis have been identified recently, but it is still completely unclear which molecules perform the fusion event itself. Although in viral fusion the fusion proteins are known, even there the molecular mechanism remains controversial. Investigation of single fusion events by electrophysiological techniques together with fluorimetric measurements have now provided some insight into the properties of the first aqueous connection, the fusion pore. This pore has an initial size similar to an ion channel and allows movement of lipids only after it has substantially expanded, indicating that it is initially not a purely lipidic structure, but incorporates lipids when it expands. Although neurotransmitter release may occur through narrow transient fusion pores, the fusion pore of synaptic vesicles probably expands vey rapidly, making it unlikely that secretion is performed by rapid exo/endocytosis without full fusion under normal conditions. Recent recordings from small membrane patches have made it possible to resolve fusion events from vesicles as small as synaptic vesicles. Future experiments using excised patches may provide an approach to identify the molecular machinery of exocytotic membrane fusion.
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Depolarization of isolated frog saccular hair cells caused Ca(2+)-dependent increases in membrane capacitance that we interpret as the fusion of synaptic vesicles with the plasma membrane. During a maintained depolarization to -10 mV, the capacitance increased at a rate corresponding to the fusion of approximately 500 vesicles per second at each active zone. Release continued at this high rate for up to 2 s, long enough to exhaust > 5 times the number of vesicles initially in close apposition to the plasma membrane at active zones. We therefore propose that hair cells are specialized for rapid replenishment of vesicles at release sites. Upon repolarization to -70 mV, the capacitance returned exponentially (time constant, approximately 14 s) to near the prestimulus level in perforated-patch recordings, but not in whole-cell recordings, suggesting that a mobile intracellular factor is required for membrane retrieval.
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In neuroendocrine cells, cytosolic Ca2+ triggers exocytosis in tens of milliseconds, yet known pathways of endocytic membrane retrieval take minutes. To test for faster retrieval mechanisms, we have triggered short bursts of exocytosis by flash photolysis of caged Ca2+, and have tracked subsequent retrieval by measuring the plasma membrane capacitance. We find that a limited amount of membrane can be retrieved with a time constant of 4 s at 21-26 degrees C, and that this occurs partially via structures larger than coated vesicles. This novel mechanism may be arrested at a late step. Incomplete retrieval structures then remain on the cell surface for minutes until the consequences of a renewed increase in cytosolic [Ca2+] disconnect them from the cell surface in < 1 s. Our results provide evidence for a rapid, triggered membrane retrieval pathway in excitable cells.
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In pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) induces the rhythmic release of Ca2+ from an inositol 1,4,5-trisphosphate (IP3)-sensitive store. Simultaneous measurement of the concentration of cytosolic free Ca2+ ([Ca2+]i) and exocytosis in single identified gonadotropes showed that each elevation of [Ca2+]i induced a burst of exocytosis. These phenomena were largely suppressed by buffering of [Ca2+]i but persisted in the absence of extracellular Ca2+. Activation of voltage-gated Ca2+ channels by brief depolarizations seldom supplied enough Ca2+ for exocytosis, but [Ca2+]i elevations induced by photolysis of caged IP3 did trigger exocytosis, confirming that GnRH-stimulated gonadotropic hormone secretion is closely coupled to intracellular Ca2+ release. Agonist-induced oscillations of [Ca2+]i in secretory cells may be a mechanism to optimize the secretory output while avoiding the toxic effects of sustained elevation of [Ca2+]i.
To study the final steps in the secretory pathway of rat pituitary melanotrophs, we have monitored changes in cell surface area due to exocytosis after flash photolysis of caged Ca2+. A step increase in cytosolic [Ca2+] to 45-125 microM triggers three phases of exocytic secretion. A small cohort of a few hundred vesicles is exocytosed in 40 ms in a secretory burst with a peak rate of 17,000 vesicles/s. Next, 2700 more vesicles are released in a slower phase that is complete within 400-1000 ms. Finally, vesicles continue to be released slowly (500 vesicles/s) for > 8s. The approach described provides a way to identify and monitor the final steps in the secretory pathway at millisecond resolution. That a small portion of secretory vesicles can be released much faster than all others suggests that these vesicles are functionally equivalent to those at the presynaptic active zone of a neuron. Their release would be fast enough to be temporally correlated with single action potentials.
Using flash photolysis of caged Ca2+ and the membrane capacitance to monitor exocytosis, we have studied the response of single melanotrophs to a step rise in cytosolic Ca2+ concentration ([Ca2+]i). Exocytosis begins with a rapid burst. This burst is followed by a slower phase, which is inhibited at cytosolic pH 6.2, and an ultraslow phase, which is strongly temperature sensitive. The exocytic burst starts with a delay of 6-11 ms and continues at a rate that grows steeply with [Ca2+]i and is half-maximal at [Ca2+]i = 27 microM. At least 3 Ca2+ ions are required to trigger exocytosis. The rate constant at saturating [Ca2+]i suggests that exocytosis of a dense core vesicle takes 40 ms after all Ca2+ ions have bound to their regulatory sites. If docked dense core vesicles cause the exocytic burst, they must decorate the plasma membrane at a mean density of 0.5/micron2.
We review here evidence that mast cells independently secrete large and small vesicles. Release of small vesicles is triggered by Ca2+. Combining capacitance measurements and flash photolysis of caged Ca2+, we demonstrate Ca-triggered exocytosis that is kinetically identical to the release of dense-core peptidergic vesicles from pituitary melanotrophs. It is suggested that the secretory pathway used for hormone secretion in neuroendocrine cells is also active in mast cells and eosinophils. The nature of the mediator thus secreted is unknown.
We have investigated the mechanism of cell fusion mediated by HA, the fusogenic hemagglutinin of the Influenza viral envelope. Single erythrocytes (RBCs) were attached to fibroblasts expressing the HA on their cell surface, and fusion of the paired cells was triggered by rapid acidification. The RBC membrane was stained with fluorescent lipid, and the fusion-induced escape of lipid into the fibroblast was observed by quantitative image analysis. At the same time, the formation of an aqueous connection (i.e., the fusion pore) between the two cells was monitored electrically. Within minutes after acidification, an electrical conductance between the two cells appeared abruptly as the fusion pore opened, and then increased gradually as the pore dilated. Later, fluorescent lipid diffused into the fibroblast, approaching equilibrium over the next 5-20 min. No lipid flux was seen while the pore conductance remained 0.5 nS or less. Evidently lipid flux requires a threshold pore size. Our finding suggests that the smallest and earliest fusion pores are surrounded by a ring of protein. A fusion pore expands by breaking this ring and recruiting lipid into its circumference.
Two new complementary DNAs overlapping cDNA clones that encode the G-protein coupled metabotropic glutamate receptor mGluR1 from rat brain have been isolated and sequenced in their entirety. These new clones represent mRNA with 3' untranslated regions approximately 2.5 kilobases longer than the previously isolated cDNA clones. These results indicate that the previously observed two size classes of approximately 4 kb and approximately 7 kb which hybridize to sequences that encode this receptor use different polyadenylation signals and differ in the extent of their 3' untranslated sequence. There is a striking asymmetry in the distribution of a sequence involved in mRNA instability between the two mRNA species.
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Tetraethylammonium ion (TEA+) blocks voltage-gated K+ channels by acting at two sites located at opposite ends of the aqueous pore. This allowed us to test two predictions made by models of ion permeation, namely that K+ channels can be simultaneously occupied by multiple ions and that the ions repel each other. We show that externally applied TEA+ antagonize block by internal TEA+ and vice versa. The antagonism is less than predicted for competitive binding, hence TEA+ may occupy both sites simultaneously. External TEA+ and internal TEA+ reduce each others affinity 4- to 5-fold. In addition, K+ antagonizes block by TEA+ at the opposite side of the membrane, and external TEA+ antagonizes is block by internal Ba2+. The antagonism between ions applied at opposite sides of the membrane may be common to all cations binding to K+ channels.