The transduction of extracellular signals into intracellular responses is mediated in part by a family of guanine nucleotide-binding proteins (G proteins) which couple receptors to effectors. However, the specific role of G proteins in organizing the communication network within a neuron is poorly understood. In the cell line NG108-15, several transmitter actions are either sensitive or insensitive to pertussis toxin (PTX). PTX blocks the coupling of receptors to, GoA, GoB, Gi1, Gi2 and Gi3. Two groups of experiments are proposed. The first involves PTX- insensitive responses. The bradykinin (BK) receptor activates a PI- phospholipase C (PI-PLC), which leads to transient activation of a Ca- dependent potassium current IK(Ca). The same transmitter also slowly inhibits the M-type potassium current (IM). Potential candidates for mediation of these events are the recently discovered PTX-insensitive G proteins, Gq and homologs. Using the whole-cell voltage-clamp technique, wild-type NG108-15 cells will be intracellulary perfused either with Gq or with an anti-Gq antibody, to see whether they simulate or block, respectively, BK modulation of these two K currents. The second group involves the PTX-sensitive responses, such as the inhibition by norepinephrine (NE) and Leu-enkephalin (Leu-EK) of the voltage-dependent Ca current (ICa,v). G(o) might to mediate these effects. However, the same receptors that activate G(o) in vitro also stimulate Gi and inhibit adenylyl cyclase. The role of G(o)s and Gis in transducing the PTX- sensitive actions of transmitters on Ica,v will be investigated. Two strategies are proposed. A novel strategy will use CDNAS encoding the alpha(o) and alpha(i) subunits which have a serine substituted for the cysteine four residues from the C-terminus (the site of ADP ribosylation by PTX). Transfected NG108-15 cell lines expressing the individual mutated alpha subunits will be isolated and the regulation of Ica,v under whole-cell voltage-clamp recording conditions examined. By recording Ica,v, in the presence of PTX, we will examine which PTX- sensitive pathway can be rescued by virtue of the expression of these mutant PTX-insensitive G proteins. Alternatively, PTX-insensitive recombinant alpha subunits will be separately perfused into PTX-treated wild-type NG108-15 cells. The PTX-sensitive transmitter actions on Ica,v, will then be investigated, to examine which pathway can be reconstituted by the exogenous G proteins.
