THE IDENTIFICATION AND LOCALIZATION OF THE GUANINE-NUCLEOTIDE BINDING-PROTEIN G0 IN THE AUDITORY-SYSTEM

The identification of guanine nucleotide binding proteins (G proteins) in guinea-pig tissues was assessed by the adenosine diphosphate-ribosylation of the alpha-subunit by Bordetella pertussis toxin using [alpha-P-32]nicotinamide adenine dinucleotide as the substrate followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Three tissues (inferior colliculus, neuroblastoma cells, and the organ of Corti) contained G0-alpha (39 kD), as well as Gi2-alpha (40 kD) and Gi1-alpha and/or Gi3-alpha (41 kD). The stria vascularis and the VIIIth nerve contained mainly Gi2-alpha, Gi1-alpha and/or Gi3-alpha, but G0-alpha was barely detectable. A purified preparation of outer hair cells from the organ of Corti contained all three pertussis toxin substrates including G0-alpha, with the Gi2-alpha (40 kD) subunit being the most prominent. The immunocytochemical localization of the G0-alpha subunit was determined by light microscopy after incubating isolated outer hair cells, Hensen cells and the stria vascularis with affinity-purified anti-G0-alpha antibodies. In hair cells a positive reaction was observed along the plasma membrane and around the perimeter of the cuticular plate (zona adherens). Positive reaction was also observed within the infracuticular network extending from the cuticular plate towards the nucleus in outer hair cells. Finally, the base of the outer hair cells also contained G0-alpha. However, it is likely that the G0-alpha that is present in this cell region is not within the hair cell itself, but rather in nerve terminals which remained attached during dissection.