Coupling of a purified goldfish brain kainate receptor with a pertussis toxin-sensitive G protein

Proc Natl Acad Sci U S A. 1992 May 1;89(9):4134-8. doi: 10.1073/pnas.89.9.4134.

Abstract

Goldfish brain has a high density of [3H]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 [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding and [gamma-32P]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, [3H]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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cattle
  • GTP-Binding Proteins / isolation & purification
  • GTP-Binding Proteins / metabolism*
  • Goldfish
  • In Vitro Techniques
  • Macromolecular Substances
  • Pertussis Toxin
  • Protein Binding
  • Receptors, Kainic Acid
  • Receptors, Neurotransmitter / isolation & purification
  • Receptors, Neurotransmitter / metabolism*
  • Virulence Factors, Bordetella / pharmacology

Substances

  • Macromolecular Substances
  • Receptors, Kainic Acid
  • Receptors, Neurotransmitter
  • Virulence Factors, Bordetella
  • Pertussis Toxin
  • GTP-Binding Proteins