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Volume 17, Number 8, Issue of April 15, 1997 pp. 2728-2737
Copyright ©1997 Society for Neuroscience

Stoichiometry and Assembly of a Recombinant GABA_A Receptor Subtype

Received Dec. 10, 1996; revised Feb. 4, 1997; accepted Feb. 6, 1997.

Verena Tretter, Noosha Ehya, Karoline Fuchs, and Werner Sieghart

Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria

GABA_A receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of , , and  subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant 132 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABA_A receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two  subunits, two  subunits, and one  subunit was derived. Similar experiments investigating the composition of 13 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with 1 and 3 subunits resulted in a stoichiometry of two  and three  subunits. Density gradient centrifugation studies indicated that combinations of 132 or 13 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of 12 or 32 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABA_A receptor assembly and support the conclusion that GABA_A receptors are pentamers in which a total of four alternating  and  subunits are connected by a  subunit.

Key words: GABA_A receptor; stoichiometry; assembly; subunit arrangement; human embryonic kidney 293 cells; chimeric subunits; density gradient centrifugation; Western blot

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