The effect of GIRK2(wv) on neurite growth, protein expression, and viability in the CNS-derived neuronal cell line, CATH.A-differentiated.

Death occurs in the homozygous mutant mouse weaver among several classes of neuron in cerebellum and ventral midbrain, because these neurons carry a mutation in the G protein-gated inwardly rectifying potassium channel, Girk2. GIRK2 is expressed in all neuronal types killed by wv in cerebellum and midbrain as well as in neurons elsewhere that suffer lesser consequences. GIRK2(wv) affects neurons postnatally, after proliferation, at the time of final differentiation. To assess the impact of GIRK2(wv) on neuronal development and viability, we introduced cDNA encoding wild-type and mutant channels into a variant of a CNS derived catecholamine cell line (Cath.a) known as Cath.a-differentiated. When cultured in serum-free medium, Cath.a-differentiated cells cease proliferation and undergo morphological differentiation, growing long neurites. Cath.a-differentiated cells do not express endogenous Girk channels. Transfection of GIRK2(wv) resulted in the death of Cath.a-differentiated cells, in a cDNA-concentration dependent manner. The highest concentration of Girk2(wv) cDNA caused loss of about half the cells, the next highest concentration one-third, and the least had no effect on viability. However, even the lowest concentration resulted in disruption of neurite outgrowth and reduced the protein products of co-transfected genes. High concentrations of MK801, which prevent Na(+) influx through the mutant channel, prevented death induced by GIRK2(wv). Cell death and disruption of neurite outgrowth were counteracted in GIRK2(wv)-expressing cells by the presence of an unrelated inwardly rectifying potassium channel, Kir2.3. These results are consistent with wv being a gain-of-function mutation, causing disruption of cellular homeostasis by mechanisms such as increased Na(+) influx and chronic depolarization which may in turn result in an excessive metabolic burden on the cell.

Girk2 expression in the ventral midbrain, cerebellum, and olfactory bulb and its relationship to the murine mutation weaver.

The mouse mutant weaver exhibits developmental deficits and cell death in several neuronal classes. weaver is almost certainly a mutation in the potassium channel, Girk2. In some vulnerable neurons, including those in the midbrain, it is not known whether weaver expression is the primary defect, or whether deficits are secondary to weaver expression elsewhere. In wild-type mice, our results point to subsets of dopamine-containing cells of the midbrain as primary targets of weaver. In the midbrain, all Girk2-positive cells examined in A9 (substantia nigra), A10, and A8 (retrorubral nucleus) are tyrosine hydroxylase-positive. The expression of Girk2 varies among and within these regions. Girk2-positive cells are most numerous in the substantia nigra, pars compacta, a region badly affected in homozygous weavers; in this region, Girk2 expression is found in cell somata and dendrites. In addition, in homozygous weavers, the remaining neuronal processes in A9 (as well as A8) are stunted. Within A10, a region largely spared in weaver homozygotes, Girk2 expression is undetectable in the most medially placed nuclei and is present in the nuclei that border A9. In the cerebellum, Girk2 immunoreactivity was also found in somata and dendrites of populations vulnerable to weaver, including the deep cerebellar nuclei. In a region not previously known to be affected, the olfactory bulb, Girk2 protein is detectable only in processes. The expression of mutated Girk2 has consequences for the olfactory bulb where ectopic cells are present in the external plexiform layer of the homozygous weaver. Our results emphasize that the Girk2 mutation may act to alter the development and maintenance of cell processes and that defects may be present in all Girk2-containing regions in weaver mutants.