COUPLING OF A PURIFIED GOLDFISH BRAIN KAINATE RECEPTOR WITH A PERTUSSIS TOXIN-SENSITIVE G-PROTEIN

Goldfish brain has a high density of [H-3]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [H-3]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [H-3]kainate binding, and kainate promotes [H-3]guanosine 5'-[beta, gamma-imido]triphosphate binding and [gamma-P-32]GTP hydrolysis. Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the beta-subunit of G proteins. When kainate receptors are purified in the presence of GTP[gamma-S], the 35-kDa protein is no longer present. Also, [H-3]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.