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Assessment of a Mutation in the HS Domain of Girk2 As a Candidate for the Weaver M U Ta Ti O N A.E

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Assessment of a Mutation in the HS Domain of Girk2 As a Candidate for the Weaver M U Ta Ti O N A.E Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH Assessment of a Mutation in the HS Domain of Girk2 as a Candidate for the weaver M u ta ti o n A.E. Mjaatvedt, 1,2 D.E. Cabin, 2 S.E. Cole, L.J. Long, G.E. Breitwieser, and R.H. Reeves 3 Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 A mutation in the GIRK2 inwardly rectifying K ÷ channel was mapped recently to the region of mouse chromosome 16 containing the wv gene and shown to occur in mutant but not in wild-type mice. We demonstrate tight linkage of the Girk2 mutation to the wv phenotype and refine the localization of the weaver (wv) gene on recombinational and physical maps. This linkage between Girk2 and ~ has existed since at least 1988 in descendants of the original mutation maintained in CS7BLI6 animals. Giric2. is shown to be transcribed in brain before the first recognized manifestation of the wv phenotype and in cultures of granule cells (GCs} isolated from cerebellum at postnatal day 8. Wild-type GCs grown in this culture system display an important developmental property--the ability to extend neurites. However, no inwardly rectifying K + current is detected in GCs cultured from either wv/~ or +/+ cerebellum under a variety of conditions that activate related channels in other tissues. This suggests that if the Gir~ mutation is responsible for the wv phenotype, it does not act by altering these electrical properties of developing GCs. The weaver (wv) mutation in mice was first char- progress to the late spermatid stage, but mature acterized in 1973 as a recessive mutation result- sperm are never released (Harrison and Roffler- ing in hypoplasia of the cerebellar internal gran- Tarlov 1993). ule layer (IGL) (Rakic and Sidman 1973). As a The best-characterized aspect of the wv phe- consequence, wv/wv mice display severe ataxia. notype is the ataxia that results from a failure of Heterozygous (+/wv) individuals are not ataxic, granule cells (GC) to mature and migrate to but develop a significantly smaller cerebellum form the cerebellar IGL. wv/wv individuals are than do control animals. The wv gene maps to a first identifiable at the day of birth (P0) by an region of mouse chromosome 16 (Chr 16) that is increased number of pyknotic nuclei in the ex- highly conserved with human chromosome 21 ternal granule layer (EGL) of the cerebellum (Sm- (HSA21), suggesting that its normal human ho- eyne and Goldowitz 1989). By 8 days of age, both molog is at dosage imbalance in Down syndrome the lack of GC migration in mutant animals and (DS) (Reeves et al. 1989). The human homolog of differences in cerebellar size and organization wv may also be involved in some forms of Par- among +/+, wv/+, and wv/wv mice are evident on kinson's disease, as wv/wv mice display a deficit histological analysis. Two distinct lines of inves- in the dopaminergic projection to the caudop- tigation suggest that the wv defect is GC specific, utamen traced to degeneration of the tyrosine but give rise to conflicting conclusions about hydroxylase-positive cells of the substantia nigra whether the defect is extrinsic or intrinsic. Aggre- beginning at postnatal day 7 (P7) (Schmidt et al. gate cultures of neurons (presumptive GC) cul- 1982). In addition to these central nervous sys- tured from the EGL of young normal mice extend tem (CNS) anomalies, sperm in the wv/wv testis neurites, whereas wv/wv aggregates do not. wv/wv cells will extend neurites when aggregated with normal cells, or when exposed to a crude 1Present address: Center for Medical and Structural Biology, +/+ membrane extract, suggesting that wv GC Medical University of South Carolina, Charleston, South Caro- lina 29403-5848. lack a factor that is essential to normal develop- 2These authors contributed equally to this study. ment and that can be supplied extrinsically by 3Corresponding author. E-MAIL [email protected]; FAX (410) 955- +/+ cells (Gao et al. 1992). However, when chi- 0461. meras are formed between +/+ and wv/wv era- 5:453-463 ©1995 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/95 $5.00 GENOME RESEARCH ~ 453 Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press MJAAWED! ET AL. bryos, wv/wv GC are never found in the IGL. not easily account for the cerebellar phenotype as This suggests that, in vivo, adjacent +/+ cells do GC are not coupled electrically at the time the wv not complement the mutation but rather that mutation exerts its effects. Patil et al. (1995) sug- the defect is intrinsic to these cells (Goldowitz gest that membrane hyperpolarization mediated and Mullen 1982; Goldowitz 1989). by the GIRK2 channel may play an important The wv gene has been localized between Cbr role in normal GC development and that this is and Pcp4, an interval amenable to positional disrupted by the mutation. cloning efforts (Mjaatvedt et al. 1993). Isolation To evaluate further Girk2 as a candidate for of genes from the corresponding region of HSA21 wv, it was mapped relative to the wv cerebellar and assessment of their murine counterparts led phenotype, demonstrating tight linkage between to the identification of a point mutation in the them. The gene was localized on a yeast artificial gene encoding the G protein-coupled, inwardly chromosome (YAC)-based physical map of the re- rectifying K÷ channel, GIRK2 (Peterson et al. gion and oriented with respect to the chromo- 1994; Patil et al. 1995). The Girk2 gene maps to some. Girk2 transcript was present by embryonic the region of Chr 16 that contains the wv gene, day 14 (E14), substantially earlier than the first and the point mutation occurs in mutant but known manifestations of the phenotype, and was not wild-type animals, altering a highly con- readily detectable in cerebellum at P0 and later. served amino acid residue in the pore-forming Cultures of purified EGL neurons from 8-day-old region of the channel. A previous in situ hybrid- mice contained Girk2 mRNA. However, no in- ization study of GIRK2 in adult mice demon- wardly rectifying K÷ current was detected in these strated distinct signals in the IGL of the cerebel- presumptive GCs, and no differences in K÷ cur- lum and in substantia nigra, the two brain areas rents were detected between +/+ and wv/wv cells known to be affected by the wv mutation, and in under a variety of conditions. Our results refine hippocampus, pontine nuclei, olfactory bulb, ce- genetic arguments for the candidacy of the Girk2 rebral cortex, septum, and amygdala (Kobayashi mutation as the basis for weaver, but suggest that et al. 1995). GIRK2 mRNA is detectable by RNase the cerebellar phenotype does not arise from dif- protection from day P1 in mouse cerebellum and ferences in inwardly rectifying K÷ currents be- in testis but not liver, kidney, lung, spleen, or tween +/+ and wv/wv GCs. heart of adult mouse (Patil et al. 1995). GIRK2 is a member of the recently identified RESULTS superfamily of inwardly rectifying K÷ channels (see Doupnik et al. 1995). Activation of this class Previously, we reported the positions of 11 genes of channels can be achieved by exposure of the mapped on a high-resolution backcross panel in cell to agonists for receptors that couple to per- the region of distal Chr 16 that is homologous to tussis toxin-sensitive G proteins, by direct persis- HSA21, and localized wv between Cbr and Pcp4 tent activation of the G proteins themselves on 104 meioses of an intersubspecific backcross through intracellular application of GTPyS, or by (Reeves et al. 1989; Mjaatvedt et al. 1993). This direct application of activated G protein subunits panel was typed with simple sequence repeat to excised membrane patches (Breitwieser 1991; (SSR) markers D16Mit52 and Mit71 (Fig. 1). Two Kurachi 1995). Pertussis toxin-sensitive G pro- crossovers between Mit52 and Cbr demonstrated teins are present in cerebellar GC as evidenced by that the SSR maps 0.34 cM proximal to Cbr (2/ the GABAB receptor-mediated inhibition of Ca 2÷ 588). Mit71 did not recombine with Pcp4 on 554 currents (Haws et al. 1993; Mintz and Bean 1993), meioses and thus is distal to wv. and therefore, signaling pathways for activation To refine the location of the wv locus, 124 of GIRK2 exist in these cells. G protein-activated progeny (248 meioses) were generated from an inwardly rectifying K÷ channels serve, in many intercross between F1 mice that were obligate cell types, to reduce excitability in a receptor- heterozygotes at the wv locus, [B6CBACa-AW-J/A- regulated manner, increasing membrane perme- wv x MOLD/Rk]F1. DNAs from these animals ability to K÷, and thus contributing to cell hyper- were analyzed with primers for Mit52 and Mit71, polarization. The Gly --4 Ser mutation within the identifying 12 crossovers. Subsequent recombi- H5 domain of Girk2 in wv mice might affect ex- national mapping of wv was accomplished using citotoxicity, which could be related to degenera- these 12 DNAs representing the informative mei- tion of neurons in the substantia nigra (Goldow- oses from among 248 generated in the intercross, itz and Smeyne 1995; Patil et al. 1995), but would plus informative DNAs from among 104 progeny 454,A GENOME RESEARCH Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press GmK2 AS A wv CANDIDATE GENE CEN detection of the mutation 128 52 119 wv, (Girk2//53) (71,Pcp4,106// 20,Erg) (Fig. 2). Twelve backcross and intercross progeny in- 2/586 1.27 110/7907 I 0"86/8/936/ N 0.91 tSlSS41 [ •, • mmm II °' II ! I cluding all informative 1.0 (11104) 1.9 (31159) 0.57 (2/352) 1.9 (4/216) 128 Cbr 119 wv Pcp4 crossovers in the vicinity of wv were evaluated to deter- / mine the parental type of the Girk2 locus.
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