Involvement of GIRK2 in postnatal development of the weaver cerebellum

We demonstrate that the homozygous weaver granule neurons cultured on a laminin substratum fail to express inwardly rectifying potassium currents, including a functional G-protein coupled inwardly rectifying potassium (GIRK)2 potassium channel. By contrast, both normal and weaver Purkinje cells express inwardly rectifying potassium currents, and normal granule cells exhibit inwardly rectifying potassium currents inducible with GTP-γ-S. In protein extracts of the vermal postnatal day (P)5-9 weaver cerebellum, the GIRK2 protein could not be detected by Western analysis, although the GIRK2 protein was detectable in extracts of the normal vermis. Northern analysis indicated that during early postnatal cerebellar development, the GIRK2 mRNA is expressed at extremely low levels being detectable at P18-23 in the normal but not yet in the homozygous weaver cerebellum. Using reverse transcriptase- polymerase chain reaction (RT-PCR), the GIRK2 mRNA was detected in both normal and weaver cerebella, but quantitative PCR confirmed that the weaver cerebellum expressed the GIRK2 gene at significantly lower levels as compared to the normal cerebellum (P = 0.01, paired t-test). Sequencing indicated that the weaver GIRK2 channel gene had the point mutation proposed to be responsible for the weaver phenotype. Rescue of both survival and neurite outgrowth of the cultured vermal weaver granule neurons by verapamil (Liesi and Wright, 1996; Liesi et al., 1999) induced expression of immunocytochemically detectable levels of the GIRK2 protein. Sequencing revealed that the GIRK2 mRNA of the rescued weaver granule neurons remained the mutated variant of the GIRK2 channel gene. Our results indicate that expression of the mutated GIRK2 protein and/or mRNA in the weaver granule neurons may be an indicator of rescue rather than death of the weaver granule neurons. That the weaver granule neurons expressed no functional GIRK2 receptors during a time period of neuronal death and migration failure suggests that the point mutation in the H5 membrane spanning region of the GIRK2 gene may associate with, but not be responsible for the weaver phenotype. (C) 2000 Wiley-Liss, Inc.