Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes: FUNCTIONAL AND STRUCTURAL PROPERTIES*

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2_flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.     

AFM observation of single,functioning ionotropic glutamate receptors reconstituted in lipid bilayers

Background

Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment.

Methods

Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate.

Results

Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements.

Conclusions

Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment.

General significance

AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.     

Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors

The ATP-gated P2X₄ receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X₄ receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X₄ receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X₄ receptors and a functional analysis revealed that P2X₄ receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X₄ receptors revealed two different forms of activated structures under 0 Ca²⁺ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X₄ receptors display pore dilation in the absence of Ca²⁺. With Ca²⁺, the P2X₄ receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X₄ receptors that correlate with pore dynamics.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Conductive polymer combined silk fiber bundle for bioelectrical signal recording

Electrode materials for recording biomedical signals, such as electrocardiography (ECG), electroencephalography (EEG) and evoked potentials data, are expected to be soft, hydrophilic and electroconductive to minimize the stress imposed on living tissue, especially during long-term monitoring. We have developed and characterized string-shaped electrodes made from conductive polymer with silk fiber bundles (thread), which offer a new biocompatible stress free interface with living tissue in both wet and dry conditions.An electroconductive polyelectrolyte, poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) was electrochemically combined with silk thread made from natural Bombyx mori. The polymer composite 280 μm thread exhibited a conductivity of 0.00117 S/cm (which corresponds to a DC resistance of 2.62 Mohm/cm). The addition of glycerol to the PEDOT-PSS silk thread improved the conductivity to 0.102 S/cm (20.6 kohm/cm). The wettability of PEDOT-PSS was controlled with glycerol, which improved its durability in water and washing cycles. The glycerol treated PEDOT-PSS silk thread showed a tensile strength of 1000 cN in both wet and dry states. Without using any electrolytes, pastes or solutions, the thread directly collects electrical signals from living tissue and transmits them through metal cables. ECG, EEG, and sensory evoked potential (SEP) signals were recorded from experimental animals by using this thread placed on the skin. PEDOT-PSS silk glycerol composite thread offers a new class of biocompatible electrodes in the field of biomedical and health promotion that does not induce stress in the subjects.     

Monitoring neural stem cell differentiation using PEDOT–PSS based MEA

Background

Transplantation is one potential clinical application of neural stem cells (NSCs). However, it is very difficult to monitor/control NSCs after transplantation and so provide effective treatment. Electrical measurement using a poly(3,4-ethylenedioxythiophene)–poly(styrenesulfonate) (PEDOT–PSS) modified microelectrode array (MEA) is a biocompatible, non-invasive, non-destructive approach to understanding cell conditions. This property makes continuous monitoring available for the evaluation/assessment of the development of cells such as NSCs.

Methods

A PEDOT–PSS modified MEA was used to monitor electrical signals during NSC development in a culture derived from rat embryo striatum in order to understand the NSC differentiation conditions.

Results

Electrical data indicated that NSCs with nerve growth factor (NGF) generate a cultured cortical neuron-like burst pattern while a random noise pattern was measured with epidermal growth factor (EGF) at 4 days in vitro (DIV) and a burst pattern was observed in both cases at 11 DIV indicating the successful monitoring of differentiation differences and developmental changes.

Conclusions

The electrical analysis of cell activity using a PEDOT–PSS modified MEA could indicate neural network formation by differentiated neurons. Changes in NSC differentiation could be monitored.

General significance

The method is based on non-invasive continuous measurement and so could prove a useful tool for the primary/preliminary evaluation of a pharmaceutical analysis. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Real-time electrochemical imaging using an individually addressable multi-channel electrode.

We developed a real-time electrochemical imaging method that uses a multiple enzyme-modified microelectrode. The method will enable the investigation of the functions of biological materials and cells. To test its effectiveness, we imaged the two-dimensional concentration distribution for hydrogen peroxide and L-glutamate in a standard solution. The multiple electrode consists of an 8 x 8 array of 30 x 30 microm2 carbon micro electrode. Each electrode was connected to a 64-channel potentiostat that could apply a potential to all electrodes at the same time. The multiple electrode was coated with an Os-polyvinylpyridine based polymer (Os-gel) containing horse radish peroxidase (HRP) to detect hydrogen peroxide, which is a very common product of oxidase enzyme. When measuring glutamate, which is a well-known neurotransmitter in the mammalian central nerve system, we modified the electrode with a bilayer of Os-gel-HRP and GluOx. The detection limit of our method was 1 microM and images of the glutamate concentration-distribution changes induced by local injection of glutamate through microcapillary were obtained in real time.     

Effect of External pH on the Cytoplasmic and Vacuolar pHs in Mung Bean Root-Tip Cells: A 31P Nuclear Magnetic Resonance Study

The effect of the external pH on the intracellular pH in mungbean (Vigna mungo (L.) Hepper) root-tip cells was investigatedwith the ³¹P nuclear magnetic resonance (NMR) method. The ³¹PNMR spectra showed three peaks caused by cytoplasmic G-6-P,cytoplasmic P_i and vacuolar P_i. The cytoplasmic and vacuolarpHs could be determined by comparing the P_i chemical shiftswith the titration curve. When the external pH was changed overa range from pH 3 to 10, the cytoplasmic pH showed smaller changesthan the vacuolar pH, suggesting that the former is regulatedmore strictly than the latter. The H⁺-ATPase inhibitor, DCCD,caused the breakdown of the mechanism that regulates the intracellularpH. H⁺-ATPase appears to have an important part in the regulationof the intracellular pH. (Received January 4, 1984; Accepted August 27, 1984)