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

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the ‘kiss-and-run’ mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.

High calcium concentrations shift the mode of exocytosis to the kiss-and-run

Synaptic transmission and cell to cell communication in the human body are frequently characterized by the release of charged transmitters and other chemical mediators from secretory vesicles or granules which then impinge on specific receptor molecules expressed on target cells1,2,3. Depending on the excitable nature, the initiating cells respond to chemical inputs by releasing vesicular granules containing specific compounds or by inducing an electrical wave such as an action potential (AP). The process of vesicle fusion with the cell plasma membrane upon stimulation and subsequent release of the granular contents (i.e. in the form of quanta) into the extracellular environment is termed exocytosis4. When measured electrochemically such release events reveal a distinctive temporal response5,6. Exocytosis recordings are also often employed to characterize the mechanism of drug action on cells. For example, amperometric recordings have shown that the Parkinson’s drug L-Dopa increases the quantal size7, i.e. the total released charge increases, a consequence of increase vesicle size. There is thus a need to develop high throughput, scalable and multi-functional electronic instrumentation in order to characterize the action of various pharmacological inhibitors, toxins and stimulants on vesicle release. Transmitter and granular release can be specifically stimulated or inhibited depending on the cell type under study. In neurons, electrical excitations in the form of action potentials (AP) propagate along the axon and stimulate neurotransmitter release in the region between the axon terminus of the pre-synaptic neuron and the dendritic spine of the post-synaptic neuron [Fig. 1(a)] called the synapse. The released transmitters impinge on specific receptors on the post-synaptic neuron exciting or inhibiting action potential generation. In immune cells such as mast cells on the contrary, exocytosis can be induced through a receptor effector function where a specific antigen-receptor interaction causes a signal cascade within the cell, culminating in the release of chemical mediators which causes an allergic response. The released compounds from mast cells impinge on cells expressing specific receptors (such as the histamine receptor on smooth muscle cells) [Fig. 1(c)] and elicit a downstream response. In this study we seek to create a CMOS bio-sensor capable of detecting granule release from mast cells as a function of transmitter-receptor induced signaling. We then extend the approach to measuring depolarization induced activity from chromaffin cells where it can function as an electronic post-synaptic sensor [Fig. 1(d)]. Such a system not only provides a test bench for fundamental exocytotic analysis by monitoring release from vesicles and action potential’s with high temporal resolution, which is paramount in understanding cellular kinetics and establishing rapid screening procedures but also sets a promising route towards future artificial synapse systems and ionic-electronic interfacing circuitry.



Figure 1

The cell-transistor synapse.



The rat basophilic leukemia cell (RBL-2H3) is a tumor cell line used frequently as an experimental model for mucosal mast cells8. The release of inflammatory mediators from mast cells is the primary event in an allergic response9. These cells serve as a robust model for understanding the underlying biophysical and biochemical mechanism which couples signals originating at the membrane receptor with a biological effector function. Immunoglobulins of the IgE class serve as antigenic receptors which are anchored to cells via the membrane protein complex FceRI. Upon stimulation with multivalent antigen, the receptors crosslink causing a signal cascade within the cell, which eventually culminates in the secretion of preformed mediators stored in the cellular granules. Mast cells form a specialized niche of the immune system, because the triggered cellular activity is immediate. Depending on the particular type of mast cells or basophil’s, secretion occurs within seconds to minutes following the IgE cross linking step. Mast cells also provide a meaningful model for cell activation by an immunological stimulus, i.e., by an antibody-antigen reaction.

We further demonstrate the device detection capability using chromaffin cells of the bovine adrenal medulla to detect neurotransmitter release and related electrical activity. The chromaffin cell is an excitable cell and allows us to study stimulus secretion coupling as mediated by both calcium entry and voltage gated channels, i.e., exocytosis induced by depolarization.

Current methods of monitoring exocytosis include fluorescent techniques1,8,10,11, carbon-fiber amperometry12 and membrane capacitance measurements13. Fluorescence techniques often require labels and sophisticated optics, which increases the complexity of the experiment. On the other hand, amperometry may be prone to noise due to low current levels involved, relies on faradaic chemistry for detection, and is challenging to miniaturize in terms of pixel density, although recent efforts have resulted in significant improvements14,15,16. Non-faradaic transistor-based measurements on the contrary extend the detection capability to electrochemically inactive molecules, are extremely sensitive to surface adsorption, record cellular signals with a high degree of temporal sensitivity, present a naturally occurring high impedance node due to the gate oxide and can render sub-cellular spatial resolution17,18,19,20 with very low input referred noise characteristics21. Previous work on transistor-based cellular sensing has primarily focused on recording electrical activity from excitable cells such cardiac myocytes22,23 and nerve cells21,24. Recently Stern and co-workers25 extended transistor based sensing to detect antigen-stimulated T-cell activation detection by CMOS-compatible semiconducting nanowire sensors, however the exact signal generating mechanism was not investigated. In another report, transistor recording of vesicle release from chromaffin cells26 was demonstrated using open-gate ISFET’s. The recorded signal was attributed solely to the change in the local pH across the double-layer interface which leads to protonation of the surface and hence a change in surface potential. In this work, we extend this understanding and demonstrate the use of CMOS compatible floating gate transistors as cell based biosensors. Specifically we demonstrate that transistors can detect degranulation from electrically non-excitable cells such as the RBL-2H3 cell line and exocytosis from chromaffin cells. In addition to extracellular pH variation, we provide preliminary evidence suggesting direct molecular binding to the sensor surface as a signal generating mechanism. Since the nature of the FET interface allows one to record both electrochemical and ionic activities simultaneously we demonstrate the detection of AP’s and vesicle release simultaneously. Furthermore, we demonstrate simultaneous quasi-static and impedimetric based signal detection suggesting that FET’s could be used to readout passive membrane properties in addition to secreted charge.

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Non-Faradaic Electrochemical Detection of Exocytosis from Mast and Chromaffin Cells Using Floating-Gate MOS Transistors

Neurotransmitter release occurs by exocytosis of synaptic vesicles triggered by Ca2+ influx through voltage-gated Ca2+ channels It was found many years ago that release shows a steep dependence on extracellular [Ca2+] and increases approximately with the fourth power of [Ca2+] ([Dodge and

/pdf/synaptotagmin-function-illuminated-3viawygz42.pdf

Synaptotagmin function illuminated.

1. A novel slowly activating voltage-dependent K+ current was observed in isolated nerve terminals from rat neurohypophysis using the whole-cell configuration of the patch-clamp technique. 2. The activation kinetics of the slow current could be fitted assuming Hodgkin--Huxley-type kinetics, an exponential, n, of 1.3 and activation time constants decreasing from 4 s at -50 mV to 0.7s at +40 mV. 3. A positive shift of reversal potential was observed when [K+] was increased in the bath solution. The current is carried mainly but not exclusively by K+ ions. 4. When intracellular free [Mg2+] was low (approximately 60 microM), average current density was 74 pA pF-1 at membrane potentials around 0 mV. In 83% of nerve terminals current amplitude was > 10 pA pF-1. 5. The slow current was never observed when the pipette contained 4.6 mM free Mg2+. At a physiological level of free Mg2+ (0.5 mM) the average current density was 16 pA pF-1. 6. When nerve terminals were analysed after patch-clamp experiments for vasopressin content by immunodetection, no difference in current amplitude was found between the terminals containing vasopressin and all analysed terminals. 7. The voltage dependence of activation was fitted by a Boltzmann equation giving a half-activation potential of -37 mV and a slope factor of about 9 mV. 8. Tail current deactivation kinetics was biexponential with time constants of 0.12 and 1.5s. Kinetics was dependent on the duration of the activating pulse. 9. Noise analysis of the slow current indicated a single-channel current of 0.33 pA at +6 mV, corresponding to a single-channel conductance of 4.3 pS. 10. This is the first demonstration of a current similar to the slow K+ current, IKs, in a neurone, suggesting that a protein similar to the IKs-inducing channel protein IsK (minK) may be present in peptidergic nerve terminals. 11. The activation properties are consistent with a role of the slow current in inhibition of excitability, at least at the level of the nerve terminal.

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A slowly activating voltage-dependent k+ current in rat pituitary nerve terminals

Stimulated exocytotic secretion from nerve endings is initiated by an increase in intracellular free calcium concentration. We summarize here our latest findings regarding the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve endings. In addition, we present surprising findings for a regulatory role of intracellular Na+ on exocytosis.

Manfred Lindau

Papers

High calcium concentrations shift the mode of exocytosis to the kiss-and-run

Non-Faradaic Electrochemical Detection of Exocytosis from Mast and Chromaffin Cells Using Floating-Gate MOS Transistors

Synaptotagmin function illuminated.

A slowly activating voltage-dependent k+ current in rat pituitary nerve terminals

Sodium, calcium and exocytosis: Confessions of calcified scientists.