Weaver Mouse Kir32 Phenotype

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Weaver mice (wv/wv) are so called because they have an ataxic gait and thus 'weave' around when they move. They also exhibit hyperactivity and tremor. These behaviours result from a selective loss of neurones in two regions of the brain, the granule layer of the cerebellum and the substantia nigra, during development. Although the precursors of the cerebellar granule cells develop normally, they fail to differentiate and migrate into the granule layer and they die during the first two weeks of post-natal life. The dopaminergic cells of the substantia nigra also die and the mice are sterile. Heterozygous animals (wv/+) possess a significantly smaller cerebellum than wild-type animals but do not exhibit ataxia.

Mutation analysis

The weaver phenotype is caused by a point mutation in the gene encoding Kir3.2, which results in substitution of a serine residue for glycine 156 (G156S), which lies within the pore loop of the channel (Fig. 8.13A; Patil et al., 1995). Mutagenesis studies, described in Chapter 6, have shown that the equivalent mutation eliminates the K+ selectivity of Shaker channels (Heginbotham et al., 1994). As was expected from these studies, when the weaver mutation was engineered into Kir3.2 the mutant channel no longer discriminated between monovalent cations (Fig.8.13B; Kofuji et al., 1996; Navarro et al., 1996; Sles-singer et al., 1996). More surprisingly, it was also constitutively open and insensitive to G-protein activation. These novel properties resulted in a large inward current through Kir3.2 channels at rest, and thus to a marked depolarization of the resting potential. Moreover, Xenopus oocytes injected with wvKir3.2 mRNA died within a few days. Oocyte death was prevented by elimination of Ca2+ from the extracellular solution, suggesting it might result

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FIGURE 8.13 THE WEAVER KIR3.2 CHANNEL IS Na+ PERMEABLE

The weaver Kir3.2 channel contains a glycine to serine mutation at position 156, which lies within the K+ selectivity filter (A) This mutation renders the channel permeable to Na+ (B-E). Currents (B,C) and current-voltage relations (B,D) were recorded from Xenopus oocytes expressing wild-type (B,C) or weaver (D,E) Kir3.2. The major cation present in the extracellular solution is indicated above each trace. From Slesinger et al. (1996).

FIGURE 8.13 THE WEAVER KIR3.2 CHANNEL IS Na+ PERMEABLE

The weaver Kir3.2 channel contains a glycine to serine mutation at position 156, which lies within the K+ selectivity filter (A) This mutation renders the channel permeable to Na+ (B-E). Currents (B,C) and current-voltage relations (B,D) were recorded from Xenopus oocytes expressing wild-type (B,C) or weaver (D,E) Kir3.2. The major cation present in the extracellular solution is indicated above each trace. From Slesinger et al. (1996).

because the membrane depolarization resulting from the inward wvKir3.2 current triggers Ca2+ entry, cell swelling and cell death.

The reason why the G156S mutation should affect the ability of G-proteins to promote channel activation remains obscure, because the G-protein binding site is thought to involve residues located in the N and C terminal domains of the channel. The simplest explanation is that binding of G^ is unaffected, but that the mechanism by which binding is translated into channel opening is impaired.

Why does the G156S mutation produce the weaver phenotype?

Studies of cerebellar granule neurones isolated from wv/wv mice revealed that they did not possess G-protein activated K+ currents, unlike their wildtype littermates (Kofuji et al., 1996). Instead, they exhibited an enhanced resting Na+ conductance and a depolarised membrane potential. These differences are explained by the altered selectivity and constitutive activation of the wvKir3.2 channel. Drugs which block wvKir3.2 currents markedly enhanced the viability of wv/wv granule cells, suggesting that Kir3.2 is not required for granule cell differentiation, but rather that it is the reduced K+ selectivity of the channel that causes cell death. Further support for this idea comes from studies of Kir3.2 knockout mice (Signorini et al., 1997). Although homozygous knockout animals did not express Kir3.2, their brains were morphologically normal and they did not exhibit ataxia. Thus it appears that increased Na+ influx, rather than loss of Kir3.2 per se, is the cause of neuronal death in weaver mice.

In wild-type animals, Kir3.2 coassembles with Kir3.1 to form heteromeric channels. What happens then, when wvKir3.2 is coexpressed with Kir3.1 in heterologous systems? It turns out that heteromeric channels show a much reduced whole-cell current, both under resting conditions and in response to G-protein stimulation (Slessinger et al., 1996). This suggests that neuronal death in wv/wv animals will only occur in those brain regions where Kir3.2 is expressed alone, and not where it is coexpressed together with Kir3.1. In support of this idea, Kir 3.2, but not Kir3.1, is expressed in those substantia nigra neurones that die in wv/wv mice. Furthermore, no structural or functional abnormalities have been reported in wv/wv animals in some of the brain regions where Kir 3.2 and Kir3.1 are coexpressed, such as cerebellar Purkinje cells, pontine nucleii, olfactory bulb, cerebral cortex, septum and amygdala. Although both Kir 3.2 and Kir3.1 are expressed in cerebellar granule cells, which die in wv/wv mice, it is possible that the Kir3.1 protein is expressed at too low a level to enable heteromerization to ameliorate the potentially toxic effects of the weaver allele.

In summary, the weaver mouse results from a gain-of-function mutation in Kir3.2. This causes a constitutive inward current that produces depolarization and cell death. The development of the weaver phenotype correlates with the neuronal cell death that follows age-dependent expression of Kir3.2. Loss of Kir3.2, as in the Kir3.2 ^ mice, does not result in the wv/wv phenotype (Signorini et al., 1997). However, these mice show sporadic stress-induced seizures and enhanced susceptibility to convulsive agents, consistent with a role for the G-protein-activated K+ current in reducing cell excitability. No human disease with symptoms matching those of weaver, or Kir3.2 knockout mice, has been reported and no disease has been mapped to the human Kir3.2 gene (which is located on chromosome 21q22.1-22.2).

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