期刊
NANOTECHNOLOGY
卷 14, 期 8, 页码 864-872出版社
IOP PUBLISHING LTD
DOI: 10.1088/0957-4484/14/8/305
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We have developed an atomic force microscopy (AFM)-based method for the determination of the subunit architecture of ionotropic receptors, and tested the method using the GABA(A) receptor as a model system. The most common form of the GABA(A) receptor probably consists of 2alpha(1)-, 2beta(2)- and 1gamma(2)-subunits. We show here that the arrangement of subunits around the central Cl- ion channel can be deduced by AFM of receptors tagged with subunit-specific antibodies. Transfection of cells with DNA encoding alpha(1)-, beta(2)- and gamma(2)-subunits resulted in the production of receptors containing all three subunits, as judged by both immunoblot analysis and the binding of [H-3]-Ro15-1788, a specific radioligand for the GABA(A) receptor. A His(6)-tag on the alpha(1)-subunit was used to purify the receptor from membrane fractions of transfected cells. After incubation with anti-His(6) immunoglobulin G, some receptors became tagged with either one or two antibody molecules. AFM analysis of complexes containing two bound antibodies showed that the most common angle between the two tags was 135degrees, close to the value of 144degrees expected if the two alpha-subunits are separated by a third subunit. This method is applicable to the complete elucidation of the subunit arrangement around the GABAA receptor rosette, and can also be applied to other ionotropic receptors.
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