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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM047721-01
Application #
3307150
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1992-05-01
Project End
1995-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Wilk-Blaszczak, M A; Stein, B; Xu, S et al. (1998) The mitogen-activated protein kinase p38-2 is necessary for the inhibition of N-type calcium current by bradykinin. J Neurosci 18:112-8
Wilk-Blaszczak, M A; Singer, W D; Quill, T et al. (1997) The monomeric G-proteins Rac1 and/or Cdc42 are required for the inhibition of voltage-dependent calcium current by bradykinin. J Neurosci 17:4094-100
Wilk-Blaszczak, M A; Singer, W D; Belardetti, F (1996) Three distinct G protein pathways mediate inhibition of neuronal calcium current by bradykinin. J Neurophysiol 76:3559-62
Horstman, D A; Ball, R; Carpenter, G (1995) Baculovirus expression and purification of the second messenger enzyme phospholipase C-gamma 1, a tyrosine kinase substrate. Protein Expr Purif 6:278-83
Wilk-Blaszczak, M A; Gutowski, S; Sternweis, P C et al. (1994) Bradykinin modulates potassium and calcium currents in neuroblastoma hybrid cells via different pertussis toxin-insensitive pathways. Neuron 12:109-16
Wilk-Blaszczak, M A; Singer, W D; Gutowski, S et al. (1994) The G protein G13 mediates inhibition of voltage-dependent calcium current by bradykinin. Neuron 13:1215-24
Kuo, W N; Dominguez, J L; Shabazz, K A et al. (1986) Modulation of the catalytic subunit of cyclic AMP-dependent protein kinase by calmodulin, S-100 protein, parvalbumin and troponin. Cytobios 46:139-46
Kuo, W N; Rahmani, M A; Blake, T et al. (1986) Probable occurrence of brain basic protein factor I, an activator of phosphoprotein phosphatases. Cytobios 47:45-52