THE ROLE OF ALPHA-1 AND ALPHA-6 SUBTYPE AMINO-TERMINAL DOMAINS IN ALLOSTERIC REGULATION OF GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS

The gamma-aminobutyric acid(A) (GABA) receptor in the mammalian centra l nervous system is composed of pentameric combinations of alpha 1-6, beta 1-4, gamma 1-3, delta 1, and/or epsilon 1 subunit subtypes. Altho ugh each of the different subunits influences the functional propertie s of gamma-aminobutyric acid(A) receptors (GABARs), the alpha subunit subtypes have been shown to be important for activation of the recepto r by GABA and pentobarbital and the regulation of GABARs by numerous a llosteric regulators, including benzodiazepines, furosemide, zinc, and lanthanum. However, with the exception of the benzodiazepines, the al pha subtype domain that is responsible for the action of these alloste ric compounds is unknown. The alpha 1 and alpha 6 subtypes are among t he most diverse of the alpha subunit family and confer a different res ponsiveness of GABARs to GABA and many of the allosteric modulators. T hese regulatory compounds act after extracellular application and ther efore likely act on extracellular GABAR sites, the largest of which is the amino-terminal extracellular domain. To determine the role of thi s domain in the action of these allosteric regulatory agents, we const ructed chimeras of the rat alpha 1 and alpha 6 subtypes with a splice site within the first putative transmembrane domain (TM). This separat ed the large extracellular amino-terminal domain from the transmembran e, intracellular, and TM2-3 and carboxyl-terminal extracellular domain s of the subunit, The chimeric subtypes were expressed in L929 fibrobl asts along with beta 3 and gamma 2L subtypes, and their pharmacologica l properties were determined with whole-cell electrophysiological reco rding. The alpha subtype amino-terminal extracellular domain was the p rimary determinant of GABA sensitivity and was responsible for the fun ctional properties of activation by pentobarbital, sensitivity to diaz epam, potentiation by lanthanum, and high affinity inhibition by furos emide. The remaining carboxyl-terminal domains influenced the GABA sen sitivity and determined zinc sensitivity and low affinity inhibition b y furosemide. Both domains were apparently required for inhibition by lanthanum.