抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

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Receptors for Purines and Pyrimidines

P2X receptors are membrane ion channels that open in response to the binding of extracellular ATP. Seven genes in vertebrates encode P2X receptor subunits, which are 40–50% identical in amino acid ...

Molecular Physiology of P2X Receptors

Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The structures of three conformations of the ectodomain of the 1968 Hong Kong influenza virus HA have been determined by X-ray crystallography: the single-chain precursor, HA0; the metastable neutral-pH conformation found on virus, and the fusion pH-induced conformation. These structures provide a framework for designing and interpreting the results of experiments on the activity of HA in receptor binding, the generation of emerging and reemerging epidemics, and membrane fusion during viral entry. Structures of HA in complex with sialic acid receptor analogs, together with binding experiments, provide details of these low-affinity interactions in terms of the sialic acid substituents recognized and the HA residues involved in recognition. Neutralizing antibody-binding sites surround the receptor-binding pocket on the membrane-distal surface of HA, and the structures of the complexes between neutralizing monoclonal Fabs and HA indicate possible neutralization mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in its structure primes HA for subsequent activation of membrane fusion at endosomal pH (Figure 1). Priming involves insertion of the fusion peptide into a charged pocket in the precursor; activation requires its extrusion towards the fusion target membrane, as the N terminus of a newly formed trimeric coiled coil, and repositioning of the C-terminal membrane anchor near the fusion peptide at the same end of a rod-shaped molecule. Comparison of this new HA conformation, which has been formed for membrane fusion, with the structures determined for other virus fusion glycoproteins suggests that these molecules are all in the fusion-activated conformation and that the juxtaposition of the membrane anchor and fusion peptide, a recurring feature, is involved in the fusion mechanism. Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) protein complex of vesicle fusion allows a similar conclusion.

Receptor Binding and Membrane Fusion in Virus Entry: The Influenza Hemagglutinin

Inositol phospholipids have long been known to have an important regulatory role in cell physiology. The repertoire of cellular processes known to be directly or indirectly controlled by this class of lipids has now dramatically expanded. Through interactions mediated by their headgroups, which can be reversibly phosphorylated to generate seven species, phosphoinositides play a fundamental part in controlling membrane-cytosol interfaces. These lipids mediate acute responses, but also act as constitutive signals that help define organelle identity. Their functions, besides classical signal transduction at the cell surface, include regulation of membrane traffic, the cytoskeleton, nuclear events and the permeability and transport functions of membranes.

Phosphoinositides in cell regulation and membrane dynamics

G-Protein regulation of eosinophil exocytosis.

Elevated intracellular Ca2+ concentrations [Ca2+] appear to be a rather universal trigger of massive membrane capacitance increases, presumably reflecting exocytosis of tiny vesicles (Borgonovo et al., 2002). The [Ca2+] required to stimulate this response is very high. In this issue Yaradanakul et al. (see p. 29) report experiments with baby hamster kidney (BHK) cells expressing the Na+/Ca2+ exchanger NCX1. In whole-cell patch clamp experiments, using an intracellular solution with high (40 mM) Na+ concentration and a Na+-free extracellular solution, switching extracellular [Ca2+] from very low (in the presence of 0.5 mM EGTA) to 2 mM is a new trick that makes it work. The massive Ca2+influx that is produced (mediated bv the Na+/Ca2+ exchanger running in reverse) raises intracellular free [Ca2+] to ∼200 μM, which triggers a pronounced capacitance increase. The response is only partially inhibited when ATP is replaced by the nonhydrolyzable analogue AMPPNP.

A Ca2+-dependent capacitance increase, distinct from secretory granule exocytosis, was first discovered in rat peritoneal mast cells (Almers and Neher, 1987) and was subsequently reported to occur in many other cell types (Lindau et al., 1993; Coorssen et al., 1996; Oberhauser et al., 1996; Xu et al., 1998; Borgonovo et al., 2002). The very high [Ca2+] increase required to induce the response in BHK cells agrees with the previously reported requirement for intracellular [Ca2+] exceeding 100 μM to induce corresponding capacitance increases in many other cells—though the response in mast cells was apparent already at ∼3 μM free intracellular [Ca2+] (Almers and Neher, 1987).

Phosphoinositides have for many years been implicated to play a significant role in regulated exocytosis (Eberhard et al., 1990; Hay et al., 1995; Martin, 2001; Wenk and De Camilli, 2004). Yaradankul et al. (2008) present detailed studies on the role of phosphoinositides in the response that is triggered by high [Ca2+] in BHK cells. The Ca2+ influx activates PI(4,5)P2 breakdown but phosphoinositide metabolism turns out to be neither sufficient nor necessary for the membrane-fusion response. Activation of PI(4,5)P2 breakdown in the absence of a sufficiently high [Ca2+] increase does not stimulate fusion and PLC inhibitors as well as peptides binding PI(4,5)P2 do not interfere with the activation of the Ca2+ influx–induced fusion response. These results indicate that the regulation of this fusion response is quite different from what has been reported for hormone release from neuroendocrine cells, where PI(4,5)P2 appears to have a role in the priming as well as the fusion (Eberhard et al., 1990; Hay et al., 1995; Martin, 2001).

What membrane compartment(s) could cause the observed massive capacitance changes? The usual assumption is that these changes reflect an increase in membrane area due to fusion of a large number of small vesicles that are not resolved as individual capacitance steps. Alternatively, for a membrane capacitance increase the plasma membrane thickness would have to decrease or its effective dielectric constant would have to increase dramatically. At present there is no evidence that such changes could occur on the required scale. The size of the response, however, could also not be explained by the vesicle numbers seen in electron micrographs (Yaradanakul et al., 2008). However, in thin sections such small vesicles might be lost, and a mechanism involving vesicle fusion still appears the most likely. Indeed, previous work identified the protein desmoyokin-AHNAK as a marker of the vesicles underlying this exocytotic response and which were named enlargosomes (Borgonovo et al., 2002).

In chromaffin cells the fusion of microvesicles that is not associated with catecholamine release is, in contrast to chromaffin granule exocytosis, not sensitive to tetanus toxin (TeTx) as well as Botulinum neurotoxins E, D, A, and C1 (Xu et al., 1998). Fusion of enlargosomes in PC12 cells is also TeTx insensitive (Kasai et al., 1999; Borgonovo et al., 2002). It would be interesting to explore if the response is also toxin insensitive in BHK cells. If that were the case, exocytosis of enlargosomes could be independent of phosphoinositide turnover.

In the second paper on the pair of articles, Wang and Hilgemann (see p. 51) extend these studies to the rat basophilic leukemia (RBL) mast cell line. Following serotonin loading, the size of secretory granules increases dramatically in RBL cells (Williams et al., 1999). Exocytosis was stimulated by addition of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Extending the recently developed method of patch amperometry (Albillos et al., 1997; Dernick et al., 2005) to giant patches, the authors demonstrate that large capacitance steps, reflecting fusion of these granules with the plasma membrane, are associated with amperometrically detected serotonin release. In addition to these discrete capacitance steps, capacitance changes that cannot be resolved as discrete steps are observed—and which are not associated with serotonin release, consistent with previous observations in peritoneal mast cells (Oberhauser et al., 1996).

The authors then proceed to explore the underlying mechanism(s) in excised giant patch capacitance measurements, which were performed on cells pretreated with latrunculin A to disrupt the cytoskeleton. Occasionally, but not reproducibly, exocytosis of large secretory granules associated with serotonin release was recorded by patch amperometry also in this configuration. The basis for the variability most likely is that when the patch is excised, the giant granules that are formed by serotonin loading do not stay docked to the cytoplasmic face of the giant patch but are left behind in the cell, tethered to the cytoskeleton.

In giant excised-patch recordings, application of high [Ca2+] from a puffer pipette stimulates massive exocytosis. These experiments were performed on patches from cells that were not preloaded with serotonin. In these cells secretory granules containing mediators such as histamine or hexosaminidase exist but they are small. It is therefore uncertain how much of the response is due to secretory granules and how much is due to microvesicles distinct from the secretory granules.

Surprisingly, the authors find almost complete inhibition of the capacitance response following TeTx treatment. Because rat synaptobrevin-1 is not toxin sensitive (Schiavo et al., 2000), the result suggests that the response is mediated by fusion of vesicles containing synaptobrevin-2 or cellubrevin. This is important because, though exocytosis of mast cell secretory granules is thought not to involve TeTx-sensitive synaptobrevin-2 or cellubrevin, recent experiments suggest that different subpopulations of vesicles undergoing regulated exocytosis in mast cells may involve different VAMP family members (Puri and Roche, 2008). The results of Wang and Hilgemann (2008) suggest that, in contrast to other cell types, exocytosis of RBL vesicles that fuse in response to high intracellular [Ca2+] is mediated by a TeTx-sensitive VAMP family member.

As for the BHK cells, vesicle fusion in RBL cells appears to be independent of phosphoinositide metabolism. However, experiments with wortmannin/adenosine, which inhibits PI(3) and PI(4) kinases, point to a possible role in priming. Using a technique that inflates cells, such that the plasma membrane detaches from the vesicles bound to the cytoskeleton (Solsona et al., 1998), Wang and Hilgemann further show that the recovery of the capacitance response after reshrinking the cells is markedly reduced following wortmannin/adenosine treatment, suggesting that phosphoinositdes may play a role in priming of these vesicles after making contact with the plasma membrane, consistent with the expected role of PI(4,5)P2 in the priming of secretory vesicles in neuroendocrine cells (Hay et al., 1995; Gong et al., 2005; Milosevic et al., 2005). It still remains to be determined what vesicle type gives rise to the large capacitance changes stimulated by [Ca2+] in the hundreds of micromolar range, and if these are at all similar for all the different cell types. The differential TeTx sensitivity in different cell types suggests a quite complex picture.

https://rupress.org/jgp/article-pdf/132/1/9/1225020/jgp_200810050.pdf

Fusion Gains Independence

Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector—carbon fiber electrode (CFE)—is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high-resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance, and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here, we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data. 

Patch Amperometry and Intracellular Patch Electrochemistry.

Among the actions of extracellular ATP is its ability to bring about the reversible permeabilization of the plasma membrane of certain cells including mast cells.’,’ The cell surface receptor involved exhibits a pattern of agonist specificities that indicate that it is distinct from all other known ATP receptor^.^ Because the nature of the pores and the mechanism of their formation is poorly understood, we have attempted to characterize them by three complementary methods, namely by observation of the uptake of fluorescent dyes, by investigation of their electrical properties under patchclamp conditions, and by detection of cell activation in response to externally applied Ca’+ and the GTP analogue GTP-y-S. The normally impermeant dyes ethidium bromide and trimethylammonium diphenylhexatriene (TMA-DPH) detect permeabilization by entering the cell and binding at sites that enhance their fluorescence efficiency. The rate of increase in fluorescence is then a measure of the degree of permeabilization. In media containing millimolar concentrations of Ca2+ and Mg2+, the permeabilizing species ATP4is present as a minor equilibrium component, and its concentration is calculated by computer program. The dependence of rate of increase of fluorescence upon ATP4 concentration is sigmoid for each dye with EGO values of 11.1 p M and 6.2 pM, respectively, and Hill coefficients close to 2. These data are in agreement with the observed EC,, of 19 pM for ATP-induced conductance changes in patch-clamped cells where the Hill coefficient is also close to 2. Cells treated with ATP in media containing divalent cations are activated to secrete by entry of Ca’+ ions. At high levels of ATP, secretion is inhibited as endogenous nucleotides are lost. This inactivation is removed when GTP-y-S is provided, showing that the cells are still capable of exocytosis. We conclude that GTP-y-S has access only at the higher levels of ATP, indicating that there is a progressive increase in the size of the pores with increasing ATP concentration.

Characteristics of ATP‐Induced Plasma Membrane Lesions in Mast Cells

Antibodies (Ab) are patterned at nanoscale precision for the precise immobilization and stimulation of immune cells. We demonstrate that the antigen bovine serum albumin (BSA) can be patterned on silicon using a photolithographically patterned polymer lift-off technique. The nanoscale pattern is realized as the polymer is mechanically peeled away in one contiguous piece in aqueous solution. Anti-BSA Ab are bound specifically to BSA to create a pattern of oriented Ab that provides a surface for eosinophil immobilization and degranulation. The patterns ranged from 0.36 /spl mu/m/sup 2/ to 4,489 /spl mu/m/sup 2/, appropriate dimensions for the 10-14 /spl mu/m diameter eosinophil cells. This method provides a new technique for immobilizing cells onto nano and micrometer scale patterns for analyzing cellular biochemical cascade events such as degranulation and studying cellular morphological changes in response to defined nanoscale antigenic stimulus.

https://ieeexplore.ieee.org/iel5/7856/21629/01002276.pdf

Manfred Lindau

Papers

G-Protein regulation of eosinophil exocytosis.

Fusion Gains Independence

Patch Amperometry and Intracellular Patch Electrochemistry.

Characteristics of ATP‐Induced Plasma Membrane Lesions in Mast Cells

Nanometer-scale antibody patterning for directed eosinophil cell immobilization and stimulation