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. 1998 Mar 17;95(6):3269-74.
doi: 10.1073/pnas.95.6.3269.

Multiple neurological abnormalities in mice deficient in the G protein Go

M Jiang  1 , M S Gold, G Boulay, K Spicher, M Peyton, P Brabet, Y Srinivasan, U Rudolph, G Ellison, L Birnbaumer
Affiliations

Multiple neurological abnormalities in mice deficient in the G protein Go

M Jiang et al. Proc Natl Acad Sci U S A. .

Abstract

The G protein Go is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, alpha2-adrenergic, M2 muscarinic, and somatostatin receptors. In vitro, Go is also activated by growth cone-associated protein of Mr 43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. To learn about the roles that Go may play in intact cells and whole body homeostasis, we disrupted the gene encoding the alpha subunits of Go in embryonic stem cells and derived Go-deficient mice. Mice with a disrupted alphao gene (alphao-/- mice) lived but had an average half-life of only about 7 weeks. No Goalpha was detectable in homogenates of alphao-/- mice by ADP-ribosylation with pertussis toxin. At the cellular level, inhibition of cardiac adenylyl cyclase by carbachol (50-55% at saturation) was unaffected, but inhibition of Ca2+ channel currents by opioid receptor agonist in dorsal root ganglion cells was decreased by 30%, and in 25% of the alphao-/- cells examined, the Ca2+ channel was activated at voltages that were 13.3 +/- 1.7 mV lower than in their counterparts. Loss of alphao was not accompanied by appearance of significant amounts of active free betagamma dimers (prepulse test). At the level of the living animal, Go-deficient mice are hyperalgesic (hot-plate test) and display a severe motor control impairment (falling from rotarods and 1-inch wide beams). In spite of this deficiency, alphao-/- mice are hyperactive and exhibit a turning behavior that has them running in circles for hours on end, both in cages and in open-field tests. Except for one, all alphao-/- mice turned only counterclockwise. These findings indicate that Go plays a major role in motor control, in motor behavior, and in pain perception and also predict involvement of Go in Ca2+ channel regulation by an unknown mechanism.

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Figures

Figure 1
Figure 1
Genomic organization of the gene encoding Go α subunits and design of the disruption. (A) Intron–exon boundaries of the cDNA coding for Goα and location of key amino acid sequences. Boxes, exons; open boxes, untranslated sequences; solid or hatched boxes, translated sequences. Exon numbering is shown below and the number of the last amino acid of each exon is shown above the cDNA. Amino acids in G1 through G5 regions are responsible for binding and hydrolysis of GTP (31). Mutations in R* and Q* of G2 and G3 reduce GTPase activity (32). Pertussis toxin (PTX) ADP ribosylates a Cys at position −4 from C terminus. (B) Genomic structure of the Goα gene (29, 30), structure of the targeting vector, 3′ and 5′ probes for Southern blot analysis, and expected restriction fragment sizes of the wild-type and the mutated alleles.
Figure 2
Figure 2
Genotype and ADP-ribosylation patterns of wild-type and αo−/− mice. (A) Southern blot analysis of XhoI-digested DNA from tail biopsies of −/− mice and +/− and +/+ littermates; the probe was the 3′ external probe shown in Fig. 1. (B) Selected tissues from wild-type mice and mice predicted by Southern blot analysis to be Go-deficient. Homogenizations were all in 27% sucrose/1 mM EDTA/10 mM Tris⋅HCl, pH 7.5. Homogenates were ADP-ribosylated without further processing in the presence of guanosine 5′-[β-thio]diphosphate, adenosine 5′-[β,γ-imido]triphosphate, 0.1% SDS, and 2 mM DTT for 30 min at 32°C in a final volume of 15 μl as described (36). NAD was then added to give 4 mM, mixed with 30 μl of 2× Laemmli’s sample buffer, and separated by urea-gradient SDS/PAGE in 9% gels (36). The gels were stained and destained over night and autoradiographed. Except for brain, the predominant bands are αi2. Note that in the pituitary the intensities of αo1 and αo2 (above and below αi2) are about equivalent; in brain, αo1 is the predominant band with αi1/3 and αi2 being similar in intensity. In heart ventricle homogenates, only the αi2 band is evident [absent in αi2-deficient hearts (37)].
Figure 3
Figure 3
Characteristics of Go-deficient mice. (A) Body weight of littermates at 3 and 8 weeks of age. (B) Survival of 33 mice αo−/− mice at 1 week old. (C) Hot-plate tests of −/−, +/−, and +/+ mice. (D) Time elapsed between start of rotation and fall off of mice from a 4.5-cm-diameter cylinder rotating at 6 rpm. For A, C, and D, data are the mean ± SEM.
Figure 4
Figure 4
Ca2+ current regulation in DRG cells from αo−/− mice and αo+/+ littermates. (A) αo+/+ cells. (Left) Lack of a depolarizing prepulse to potentiate Ca2+ channel current elicited by a test pulse to −5 mV. (Right) Reversal by prepulse of opioid receptor-mediated inhibition of Ca2+ channel current. (B) αo−/− cells. (Left and Right) As in A. (Inset) Voltage protocol used in A and B. (C) Means of opioid inhibition of Ca2+ channel current in αo−/− and αo+/+ cells. Inhibition by DAMGO was 39 ± 3.6% for +/+ and 27.6 ± 3.8 for −/− cells. The difference, 11.4 ± 5.2%, is significant at the P < 0.05 level. (D) Current–voltage relationship of Ca2+ channel current in αo+/+ (○) and αo−/− cells (▴, occluded by circles, and ▪). Thirty-one αo−/− and 21 αo+/+ cells were analyzed. Current–voltage curves of αo−/− cells fell into two groups: one with a V1/2 of activation of −26 ± 1.5 mV and the other with V1/2 of −12.7 ± 0.8 (mean ± SEM; P < 0.001). V1/2 in +/+ cells was −14.2 ± 0.9 mV.
Figure 5
Figure 5
Turning behavior of αo−/− mice. Results from an open-field test in which a mouse is placed in a 13 × 17 inch box with a flat bottom covered with a 1-inch layer of sand (for details, see text). The lower superpositions show the movements of five αo−/− mice (2065M, 2093F, 2155M, 2370M, and 2371F, where M is male and F is female). 2065M and 2371F walked almost exclusively along the sides of the box.
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