2012-2013 ARDRAF Project

Identification and characterization of nAChRs clustered in cell membrane lipid rafts using novel patching technique with chemically modified electrodes

VCoA-at-VCU*The Virginia Center on Aging which administers the Alzheimer’s and Related Diseases Research Award Fund for the Commonwealth of Virginia, provides seed money to researchers in Virginia to stimulate innovative research into biomedical and psychosocial aspects of dementia, including cell biology, caregiving, and animal modeling.


 

Identification and characterization of nAChRs clustered in cell membrane lipid rafts using novel patching technique with chemically modified electrodes

Vladimir Sidorov, PhD

Virginia Commonwealth University 

Recently, it has been shown that the function of alpha4beta2 and alpha7 subtypes of nicotinic acetylcholine receptors (nAChRs) is regulated by association of the receptors with rigid areas of neuronal membranes, known as lipid rafts. The only currently available experimental tool to study receptor channel regulation by the cell membrane lipid rafts entails modifying the interaction with methyl-beta-cyclodextrin. The overall goal of this project is to develop a novel technique that allows identification and characterization of the functional properties of nAChRs based on selective patching of the raft and fluid areas of cell membranes with a chemically modified borosilicate electrode. This approach will serve as a valuable tool to monitor the activity of nAChRs in the specific membrane areas, including their regulation by beta amyloid, one of the major players in the development of AD. The initial stage of this investigation is aimed at the development of a procedure for surface modification of the borosilicate electrodes used in the patch-clamp recordings with the chelator for pyranine dye, and labeling of the cell membrane domains with pyranine conjugates. The mass-spectral analysis of the contents of patches will allow the investigator to establish feasibility of selective patch-clamp recordings for the raft and fluid membrane areas. As additional funding becomes available, the next step will compare the properties and sensitivity to beta amyloid of alpha4beta2 and alpha7 nAChRs expressed in the lipid rafts and free cell membrane regions. The completion of this project will directly lead to
understanding of the effects of beta amyloid on the nicotinic receptors at the subcellular level, and may lead to new therapeutic strategies for the management of AD.

(Dr. Sidorov may be contacted at 804/828-7507; vasidorov@vcu.edu)

REPORT:

Neuronal nicotinic acetylcholine receptors (nAChRs) are critical to cell functioning and essential in the development of Alzheimer’s disease. The function of alpha4beta2 and alpha7 subtypes of nAChRs is regulated by association of the
receptors with rigid areas of neuronal membranes, known as lipid rafts. The overall goal of this project is to develop a novel technique that allows identification and characterization of the functional properties of nAChRs based on selective patching of the raft and fluid areas of cell membranes with a chemically modified borosilicate electrode. During this initial stage of the investigation, a robust chemical procedure for surface modification of borosilicate electrodes was developed for use in the planned electrophysiological experiments. The procedure allows tethering of a synthetic macromolecule, cyclen 2, which serves as a selective binding agent for the fluorescent dye pyranine. The binding
phenomenon is readily observed due to the fluorescence quenching. In order to utilize these electrodes, a series of lipid conjugates with pyranine have been synthesized. The confocal microscopy imaging experiments revealed that the
cholesterol-pyranine conjugates rapidly partition into the dynamic areas of cell membranes consistent with the ordered domains (rafts). While being in a rapid lateral motion, the fluorescently labeled cholesterol remained fully accessible for interactions with cyclen 2 attached to the glass surface. Future undertakings will test the hypothesis that the functional behavior of nAChRs is directly affected by their localization in the ordered membrane domains. Such characterization of nAChRs docked in the raft areas of membranes may lead to better understanding of key factors in the development of Alzheimer’s disease as well as to the methods for treatment of this condition.

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