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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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-

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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

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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. In each 128 119,53, Girk2,Mmb 20,95 106,0cp4 0.97 [ 0.64 case, the Girk2 type was the E~E3 mm • I I °'32 I , = • same as wv (i.e., no recom- Figure 1 Recombinational map of the wv region of Chr16 (top). Recombina- bination was seen between tion fractions are shown in each interval for all crosses (above the chromosome) the mutation and the phe- and for the wv crosses (below the chromosome). No recombination was seen notype on the informative between wv, Girk2, and Mit53, nor between Mit71, Mit106, Mit20, Pcp4, and Erg. DNAs from a total of 352 However, physical mapping data divides and orients each of these clusters as indicated by//. Orientation relative to the Chr 16 centromere (CEN) and the meioses). DNAs prepared in position of Cbr are shown. The EUCIB map (drawn to the same scale) (bottom) 1988 from a +/wv and a wv/ with the positions of Girk2, Mmb, and Pcp4 (Patil et al. 1995) is included for wv mouse that carried the comparison. wv allele on the original C57BL/6-derived strain (Rakic and Sidman 1973; Reeves et al. 1989), also of the previously characterized backcross. The showed the expected type at the Girk2 locus. map was refined using DNAs from 490-584 prog- Analysis of these animals was repeated using sin- eny of three additional intersubspecific back- gle-strand conformation polymorphism analysis crosses (Table 1). This panel was typed to gener- (SSCP) (Fig. 2) and extended to type an additional ate the map, Mit52-Cbr-119-wv, Mit53-Mit71 16 animals (32 alleles) of known wv phenotype (Fig. 1). No recombination was seen between wv for the mutant Girk2 allele, detecting no recom- and Mit53 on the mapping panel. bination. Twenty-three additional +/+ mice from Primers specific for Girk2 (Table 2) were de- a variety of strains were tested, including A, CBA, veloped based on human and mouse Girk2 se- and C57BL/6; the wv mutation arose on C57BL/6, quences (Lesage 1994; Kobayashi 1995; Tsaur and is maintained on a [CBA x A]F1 hybrid back- 1995). The GIRK2-TM primer set amplified a 263- ground at the Jackson Laboratory, the main sup- bp product within the exon that encodes the M2 plier of wv mice. None of these animals showed putative membrane-spanning domain and H5 variation at the Girk2 locus. pore-forming region and includes the base mu- Five mouse-derived YACs were identified tated in wv mice (Patil et al. 1995). The forward with the GIRK-TM primers. All were positive for primer of this set recognizes a region that is di- Mit53. Two were analyzed in detail to prepare a vergent between known members of this subfam- physical map of the region (Fig. 3). Nested dele- ily of inwardly rectifying K+ channels (Lesage et tion derivatives were prepared from YACs I6B12 al. 1994; Doupnik et al. 1995). DNAs from seven and I90E3 using centric and acentric Bl-targeted animals known to be +/% +/wv, or wv/wv were fragmentation vectors, respectively. Parental assessed by sequencing to validate this assay for YACs and derivatives were analyzed for Mit53,

Table 1. Crosses Used in This Study--Female Parent is Indicated First in the Backcrosses

Cross Reference

BALB/cJ × (BALB/cJ × MOLD/Rk)F 1 (Reeves et al. 1990, 1991 ) (BALB/cJ × MOLD/Rk)F 1 × BALB/cJ (Reeves et al. 1990, 1991 ) (MOLD/Rk × DW/J)F 1 × DW/J (Camper et al. 1990) C57BL/6 wv/wv × (MOLD/Rk x C57BL/6 wv/wv) (Reeves et al. 1989; Mjaatvedt et al. 1993) Intercross between (B6CBACa-AW-J/A-wv × MOLD/Rk)F1 mice (this study)

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MJAATVEDT ET AL.

These subdivide the cluster Table 2. PCR Primers and Reaction Conditions into a 130-kb segment containing pA33 and GIRK2-3', Name Sequence Position Size which is separated by 10 kb la. GIRK2-TM F 5' GGA CCA CAT AGA GGA CCC base 369a 263bp from an 80-kb segment de- limiting GIRK2-TM and lb. GIRK2-TM Rb 5' GCA TGG GTG GAA AAG ACC base 632 Mit53. YAC I90E3 overlaps at its centric end with YACs 2a. GIRK2-3' F 5' GGA GAG GAA TGG GTG ATG base 1230a 255bpc in a contig that extends proximally beyond Cbr (D. 2b. GIRK2-3' R 5' CAG GCC GTC TGC AAG AAC base 1485 Cabin, J. McKee-Johnson, M. Citron, and R. Reeves, in 3a. mJ3actinF 5' GGA GAA GAG CTA TGA GCT GC base 630d 374bp prep.) and thus, the Girk2 gene is oriented on Chr 16 3b. ml3actinR 5' CCT GCT TGC TGA TCC ACA TC base 1003 with the 3' end closest to the Conditions: Primer pair 1 : 30 sec at 94°C, 30 sec at 55°C, 1 min at 72°C for 45 cycles; 2 centromere. The physical mM MgCI 2. Primer pair 2:45 sec at 94°C, 45 sec at 50°C, 1-min at 72°C for 40 cycles; 1.5 map confirms the order mM MgCI 2. Primer pair 3:1 min at 94°C, 1 min at 60°C, 1 min at 72°C for 30 cycles; 3 mM Cbr-+ 119 --+ Girk2 deter- MgCI2. mined on the recombina- aNucleotide positions are from the coding sequence of mouse GIRK2 (Lesage et al. 1994). tional mapping panel. bprimer pair 1 was developed from a human sequence-tagged site (GenBank accession no. G02354; Tsaur et al. 1995). The third base of the R primer is a G in mouse, but the primers amplify mouse DNA despite the mismatch. Cprimer pair 2 produces a product of the expected size only from reverse-transcribed RNA Temporal Pattern of Girk2 template, and not from genomic DNA. Expression dpositions are from the sequence of mouse J3-actin cDNA (GenBank accession no. X03765). The Girk2-3" primers amplify the expected 255-bp product from cDNA and do the GIRK2-TM and GIRK2-3' PCR products, and not amplify genomic DNA. These primers were pA33, the acentric end-clone of a third Girk2- used to analyze Girk2 transcripts in brain from positive YAC, 182A2. A 105-kb and a 115-kb de- El0 through P30 by RT-PCR (Fig. 4). Girk2 tran- rivative of YAC I90E3 lacked all four markers, script is present in RNA extracts from whole brain whereas all were retained on a 310-kb derivative, by E14. Expression was readily detected in cere- placing them within 195 kb of each other. Figure bellum at P0, the earliest point at which wv/wv 3 shows four fragmentation derivatives of I6B12. mice can be distinguished from nonmutant animals (Smeyne and Goldow- itz 1989). Thus, the gene is + ~ transcribed in cerebellum at wv/wv +/wv +/+ "g "~ the time that the pheno- A G C T AGCT ACGTAGCT type is detectable. Tran- m script was also detected in •~" G GT G + testis and pancreas as de- •" GTA G m scribed previously (Lesage C/T ~'CCAA et al. 1994; Patil et al. 1995; ~T C/T "" Tsaur et al. 1995).

Figure 2 Detection of the Girk2 mutation at position 466 by sequencing and Absence of Inwardly SSCP. Results of sequencing products from wv/wv, +/wv, and +/+ animals are Rectifying K ÷ Current in shown. Using the GIRK2-TMR oligonucleotide as a sequencing primer, the wild- +/+ and ~/~ Cultured type sequence is C at position 466, and the mutant allele is T (Patil et al. 1995). The last panel shows an SSCP analysis of +/wv, +/+, and wv/wv mice. A "+" Cerebellar Neurons indicates bands corresponding to the wild-type allele (i.e., a C at position 466) and "m" indicates bands specific to animals with the Girk2 mutation (T at Presumptive GC were iso- position 466). A nonspecific band appears in all lanes just below the highest lated from the cerebellum mutant specific band. of mice at P8 and assessed

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GmK2 AS A wv CANDIDATE GENE

pA33, GIRK 3' GIRK-TM, Mit53 in K+ current were elicited by 1 mM pipet + + 10 GTPTS in 5 cells from wv/wv and 10 cells 105 I I 195 kb 1 + + 440 kb 190E3 310 from +/wv animals tested under similar 115 105 conditions. The presence in these cells + + of functional, pertussis toxin-sensitive G

16B12 O'1 13okb 1o 8o I 650kb proteins was confirmed by GTPTS- '' + +-I- 520640 mediated decreases in inward currents 4- 510 in the presence of 50 mM extracellular 430 BaC12 (data not shown). Cells were mon- Figure 3 Fragmentation derivatives of Girk2-positive YACs 190E3 and 16B12 were typed to determine the orientation of pA33, Girk2, itored for GTPyS-mediated changes in and Mit53. GIRK2-TM and GIRK2-3' refer to the presence of the current for an average of 15-30 rain, and PCR products corresponding to these primers (Table 2). The pres- several cells were monitored for 45 rain. ence (+) or absence (-) of pA33/GIRK3' and GIRK-TM/Mit53 No changes in outwardly rectifying K+ groups on each derivative are indicated. Sizes of the YACs and currents were observed, nor was there derivatives are in kilobases (kb). The Chr 16 centromere is to the left development of additional, inwardly (i.e., the same orientation as Fig. 1). rectifying currents. Control cells were also examined with a pipet solution containing 0 ATP, 0 GTP (n = 7), but for structural and functional integrity (Gao and no inward currents indicative of the presence of Hatten 1993). Identity of the cells was established KATP channels were elicited. In two experiments, by histochemical reactivity of >95% of cells with 150 ~JM pinacidil, which activates KATP channels tetanus toxin (not shown) and with antibody (Tseng and Hoffman 1990; Zegarra-Moran and Q600, which is specific for GC (Gravel et al. Moran 1994), failed to affect cellular K+ currents 1987) (Fig. 5). Biological integrity of the cells was in GCs. demonstrated by showing that aggregate cultures extend neurites (Fig. 5E). RT-PCR analysis of +/+ and +/wv cultures demonstrated the presence of Girk2 mRNA (Fig. 5F). E GCs were examined by the whole cell patch < .c: ,-.-.c_ ,.- E .o clamp technique. Outward K+ currents were ob- z ,~ ,~ ~ ~ = served in +/+ GCs at potentials more positive ,..-, ~.a.a.a ~ 8 •- ~ -o than -30 mV and were comprised of a transient E .~ o.~ o ~c outward current (IA) and a delayed current (IK) as o - described previously for rat GCs (Jalonen et al. T UJ W WW WW UJW W ~_ ,_ 1990; Galdzicki et al. 1991; Zegarra-Moran and Moran 1994). Analysis of GCs from wv/wv mice yielded comparable results (Fig. 6B). The normal- 3' GIRK2 ized, averaged current/voltage relationships for six +/+ and five wv/wv GCs are illustrated in Fig- 255 bp - ure 6C. wv/wv GCs displayed a somewhat higher half-maximal activation voltage for the outward 13 actin currents, which might represent slight differ- 374 bp - ences in the relative expression of Ia and IK. HOW- ever, the linear, inward currents were identical in Figure 4 Developmental and tissue profile of +/+ and wv/wv GCs. These inward currents ex- Girk2 expression. Staged embryos were isolated trapolate through 0 mV and most likely represent from timed matings. RNA was isolated from whole contributions from both leak and CI- currents. head at E10, whole brain on E14-E16, from the an- terior or posterior half of the brain at later prenatal Substitution of pipet K+ with N-methyl-D- time points, and from isolated cerebellum at P1 and glucamine (NMDG) abolished all outward cur- P30. For all analyses, RT-PCR analysis was per- rents, confirming that K+ channels were respon- formed with the GIRK2-3' primers that identify a sible. No changes in currents were observed in 255-bp product in cDNA but not genomic DNA us- response to 10 gM of baclofen (n = 2), 10 gM of ing conditions described in Table 2. Amplification dopamine (n = 3), or 10 gM of acetylcholine with ]3-actin was used as a positive control for the (n = 3). Furthermore, no time-dependent changes presence of cDNA.

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MJAATVEDT El AL.

f alter the timing of expression during develop-

#l ment, or change the cell or tissue distribution of

:.'," transcription. Third, candidacy of a mutation can be strengthened by demonstration of a plausible mechanism by which the candidate mutation could produce the phenotype. Finally, direct proof of the role of the candidate gene is obtained by the demonstration of an indepen- dently occurring mutant allele that results in the same phenotype, by the demonstration that the mutant phenotype is complemented by a normal copy of the gene in transgenic mice, or by reca- pitulation of the mutation via gene replacement. The Girk2 mutation meets several criteria as a candidate for wv. It was mapped initially between D16Mit128 and Pcp4 to the same locus as Mit53 and Mit119 in an interval of Chr 16 measuring -1.9 cM on an interspecific cross (Patil et al. 1995). This result does not formally restrict Girk2 to the previously defined wv candidate region that is delimited proximally by Cbr, as Mit128 is proximal to Cbr. Interspecific maps are com- pressed in this region (Reeves et al. 1990). On the crosses segregating wv in this study, the interval between Mit128 and Pcp4 was 5.4 cM or >5% of E F the recombinational map of Chr 16, a region that Figure 5 Cell type specificity and biological integ- might be expected to contain >150 candidate rity of GC cultures. More than 95% of cells reacted with GC-specific Q600 antibody; cultures are pic- genes. The localization of both Girk2 and wv was tured under fluorescence (A) and phase (B). Note refined using a high resolution mapping panel presence of one Q600 negative cell. No staining and YAC-based physical mapping. Mit119 was was observed with secondary antibody alone mapped 0.57 cM proximal to wv and Girk2 on our viewed under fluorescence (C) and phase (D). GC high resolution panel, whereas physical mapping extend neurites when cultured as aggregates (E). placed Mit53 distal to the 3' end of the gene, and Girk2 transcript was detected by RT-PCR with the <80 kb from the transmembrane-encoding por- GIRK2-3' primers in adult cerebellum and in GC tion of Girk2. No recombination was seen be- cultures from +/+ and +/wv animals (F). (A-D) Bar, tween the G -~ A mutation and the wv cerebellar 25 ~m; (E) bar, 10 ~m. phenotype on >350 chromosomes, providing a 95% probability that the genes are separated by <0.8 cM. This segment will be delimited further DISCUSSION as the remaining six crossovers between wv and the closest flanking markers are positioned pre- Several criteria must be met to determine cisely. whether a candidate gene is responsible for a spe- GIRK2 mRNA is detectable in brain at E14, cific mutation. First, the candidate should map to several days before the wv phenotype is evident, the correct chromosomal region and show no re- and is expressed in cerebellum throughout late combination with the mutant phenotype in a embryonic and postnatal development. Message significant number of affected individuals (as- is detected in testis, as might be expected, as wv/ suming complete penetrance). Second, the mu- wv males are known to have a defect in spermato- tant allele should differ from wild type in struc- genesis. Expression also occurs in pancreas (Tsaur ture and expression. Frequently, structural differ- et al. 1995) and in multiple brain regions not ences can be identified by analysis at the DNA or known to be affected by the wv mutation (Koba- protein level. Changes in expression can be man- yashi et al. 1995). Ultimately, the role of this mu- ifested in several ways. The mutation could result tation should be considered at all sites where the in different levels of expression (up or down), gene is expressed.

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GleK2 AS A wv CANDIDATE GENE A

120

100 80 C 60 0) p. 40 T 8 20 0 hl .20 ¢0 4O O z -60 B -8O I | I I 1 I I I I I -100 -1~ -120 -100 -80 -60 -40 -20 0 20 40 60 80 100

Membrane potential (mV)

Figure 6 Electrophysiological characteristics of mouse cerebellar GC. Voltage clamp current responses from +/+ (A) and wv/wv (B) GC held at -85 mV and stepped to potentials from -125 mV to +105 mV in 10-mV increments, with a return to holding potential between voltage steps. Pipet solution contained 1 mM GTPyS. Traces were taken -15 min after patch rupture. Bars, 200 pA and 50 msec. (C) Steady-state currents (measured at 250 msec after initiation of the pulse) were normalized to the steady-state current obtained at +75 mV. Normalized currents were averaged for six +/+ and five wv/wv GC and the current-voltage relationships plotted. Average steady-state currents at +75 mV were 98.5 _+ 39.4 pA for +/+ and 84 _+ 35 pA for wv/wv cells. (e) +/+GC; (©) wv/wv GC.

Neurons cultured from mouse cerebellum GTPyS (Haws et al. 1993). In similar experiments during the first 2 weeks after birth show many in atrial myocytes, 1 mM pipet GTPyS elicits max- properties of GCs, including the ability to partic- imal activation of muscarinic K+ currents within ipate in migration and IGL formation when in- 3-S min (Breitwieser and Szabo 1988). The ab- jected into the cerebellum of newborn mice (Gao sence of inwardly rectifying K+ channels in cul- et al. 1992; Gao and Hatten 1993). Electrophysi- tured mouse GCs 8 days after birth is consistent ological properties of these presumptive GC pre- with previous observations of rat GCs (Jalonen et cursors were examined to identify possible func- al. 1990; Galdzicki et al. 1991; Zegarra-Moran tional consequences of the Girk2 mutation. Our and Moran 1994). observations do not demonstrate the presence of Girk2 transcript was present in the GC cul- inwardly rectifying K÷ channel currents in these tures as determined by a robust signal using RT- cells under a variety of conditions that stimulate PCR (Fig. SF). Thus, the Girk2 gene is transcribed other members of this gene family. Neither ba- in at least some cells in these cultures that are clofen nor dopamine elicits changes in net mem- composed of >95% GCs, but its expression does brane currents in normal or mutant GCs. Fur- not result in the establishment of inwardly recti- thermore, inclusion of 1 mM GTPyS in the pipet fying currents analogous to those in other cells. solution did not elicit activation of an inwardly Although cerebellar development is distinctly af- rectifying K÷ current over 15- to 30-min periods fected in both +/wv and wv/wv mice, no differ- (Fig. 6) (n = 17 control; n = 16 wv/wv and +/wv), ences were seen in the electrophysiological prop- although persistent activation of pertussis toxin- erties of +/+, +lwv, or wv/wv GCs. The culture sensitive G proteins in mouse cerebellar GCs is model used here contains the potential for isola- complete in 5 rain upon exposure to 500 gM tion-induced alterations in GC properties, partic-

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MJAATVEDT ET AL. ularly to the signal transduction pathways lead- of transgenic or gene replacement mice that ing to GIRK2 activation. This problem is circum- should demonstrate directly whether the Girk2 vented by applying GTPyS intracellularly to mutation is responsible for the wv phenotype. activate G proteins directly, which are localized to the cytoplasmic face of the plasma membrane, thus bypassing extracellular receptors that could METHODS be affected by the cell isolation procedure. The Genetic Mapping muscarinic K÷ channel comprised of GIRK1 and CIR is resistant to extracellular collagenase, Animals carrying the wv mutation were obtained from the trypsin, and protease, which are used typically to Jackson Laboratory, where they are maintained on an F 1 hybrid genetic background, B6CBACa-AW-J/A-wv. The four dissociate myocytes acutely for same day record- backcrosses used in this analysis have been described pre- ing (Tung and Morad 1985; Fischmeister and viously (Table 1). Additional meioses informative for wv Hartzell 1986). As demonstrated in rat and mouse were generated in an intercross. Homozygous wv (wv/wv) GC, a variety of signaling pathways that regulate females were crossed to males of the inbred Mus musculus Ca 2+ channels are robustly present after isolation subsp, molossinus strain, MOLD/Rk, and an intercross established between their obligate +/wv progeny. One hun- (Marchetti et al. 1991; Haws et al. 1993; Huston dred twenty-four F2 progeny were sacrificed between P6 et al. 1993; Lambert and Feltz 1995), including and P21. Whole brains, liver, kidney, and spleen were im- those using pertussis toxin-sensitive G proteins mediately frozen in dry ice and stored at -80°C. DNA and that also interact with Girk2. Recent results sug- RNA extraction, Southern blotting, radiolabeled probe gest that GIRK channels must heteromultimerize synthesis, and hybridization were accomplished using standard procedures as described (O'Hara et al. 1988, 1989; with other members of the subfamily to produce Reeves et al. 1991). Clone pCR311 was used to detect an functional channels (Ferrer et al. 1995; Krapvin- EcoRI restriction fragment length polymorphism (RFLP) at sky et al. 1995; Nichols et al. 1995), and thus, the Cbr (Mjaatvedt et al. 1993). PCR primers for D16Mit mark- presence of GIRK2 alone may not be sufficient to ers were obtained from Research Genetics, Inc.; additional ensure functional channel expression. It remains primers and reaction conditions used in all experiments are described in Table 2. PCR products were separated on to be seen whether GC cells express other mem- 3.5% low melting point agarose gels and visualized under bers of the GIRK family during the postnatal time UV light after staining with ethidium bromide (Irving et al. period under investigation in this study. 1993). The wv mutation arose in the inbred SSCP was carried out as described (Orita et al. 1989), C57BL/6 strain in 1973 (Rakic and Sidman 1973). using PCR products prepared with y-:~2p-end-labeled GIRK2-TMF and/or GIRK2-TMR primers to the pore- wv/wv +/wv DNA isolated in 1988 from and de- forming region of Girk2, which contains the G -9 A muta- scendants of the original line maintained on the tion at position 466 of coding sequence (Lesage et al. 1994) B6 background carried the Girk2 point mutation, (Table 2). Products were denatured, then resolved on non- suggesting that it has been maintained with wv denaturing 6% acrylamide gels containing 5% or 10% for at least 20 generations. Given the tight link- glycerol run at 6 W for 14 hr at room temperature. Ampli- fied PCR products were sequenced with the Sequenase sys- age of Girk2 with wv, it would be expected to tem (U.S. Biochemical) using the GIRK2-TMF primer (0- to cosegregate in animals selected for inheritance of l-rain extension) and the GIRK2-TMR primer (2-rain ex- the wv phenotype. Initial experiments to assess a tension) and resolved on 6% acrylamide denaturing gels. possible functional role for the Gly --4 Ser muta- Products from at least two independent PCR reactions of tion in GIRK2 function that might account for every DNA were sequenced in both directions. The weaver genotype was assessed by histological ex- the cerebellar phenotype do not demonstrate in- amination of cerebella. Frozen brains were cut parasagi- wardly rectifying K÷ currents in cultured GC from tally and half of the brain was embedded in optimally either +/+ or wv/wv mice at P8. controlled temperature (OCT) tissue freezing medium The genetic evidence provided here strength- (Sigma Chemical) for cryostat sectioning. Midline parasag- ens the likelihood that the Girk2 mutation is re- ittal sections were cut at 10-gm thickness and stained with cresyl violet using standard techniques. RT-PCR was per- sponsible for the wv phenotype. The impact of formed on RNAs harvested from indicated tissues using this mutation on noncerebellar aspects of wv, Superscript reverse transcriptase according the manufac- (i.e., the spermatogenesis defect, seizures, and the turer's instructions (Life Technologies, Inc.). presence or absence of a phenotype in tissues that express GIRK2 but are not known to be affected) remains to be assessed. The high- Physical Mapping on YACs resolution physical and recombinational map- Mouse-derived YACs were identified by PCR-based screen- ping greatly delimit the possible location of wv ing of Research Genetics, Inc. pools of the Massachusetts candidates and provide reagents for the creation Institute of Technology library and the Baylor School of

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GIRK2 AS A wv CANDIDATE GENE

Medicine pools of Imperial Cancer Research Foundation nant TFC fragment as described in the manufacturer's in- (ICRF) and St. Mary's libraries. Standard conditions were structions, washed in PBS and incubated with anti-TTC used for preparation of high molecular weight DNA in aga- monoclonal antibody (1:200; Boehringer Mannheim) for 1 rose plugs (Pavan et al. 1990). DNA for PCR and conven- hr at 20°C. The remainder of the staining procedure was tional Southern blot analysis was prepared by the method identical to that for Q600. Staining with secondary serum of Hoffman and Winston (1987). Physical mapping of alone was used as a negative control. markers on YACs was facilitated by YAC fragmentation (Pavan et al. 1990) using a centric YAC fragmentation vector pLM1 (L. Matesic and R. Reeves, in prep.) and an acen- Electrophysiology tric vector, pJW522 (kindly provided by J. Edmundson, Rockefeller University, New York) that target homologous Whole cell currents were recorded by standard high reso- recombination to murid B1 repetitive elements. Frag- lution patch clamp techniques as described previously mented YACs were analyzed using PCR products of Girk2 (Scherer and Breitwieser 1990), with an LIST EPC-7 ampli- and pA33 as hybridization probes. fier, filtered at 2 kHz (8-pole Bessel filter) and stored on computer and/or VCR tape. Data acquisition and analysis was performed with Pclamp software (version 5.5.1; Axon GC Preparation Instruments). All experiments were performed at a holding potential of -85 mV and at 20°C-22°C. The bath solution GCs were isolated as described (Huettner and Baughman contained 150 mM NaC1; 5 mM KC1; 2 mM MgC12; 2 mM 1986; Gao et al. 1992). Briefly, cerebella from P2 to P8 mice CaC12; and 10 mM HEPES (pH 7.3). The pipet solution con- were dissected in calcium- and magnesium-free phos- tained 160 mM KC1; 2 mM MgCl2; 2 mM KEGTA; and 10 mM phate-buffered saline (CMF-PBS; GIBCO-BRL) to remove HEPES (pH 7.2). In certain experiments, the pipet solution meninges. The remaining tissue was minced and placed in was supplemented with 1 mM GTPyS (Li salt), 5 mM Earl's balanced salt solution (EBSS) containing 20 U/ml of MgATP. papain and 0.005% DNase (Worthington Biochemical Corporation). The tissue was incubated at 37°C for 1 hr then triturated and centrifuged at 70g through a gradient ACKNOWLEDGMENTS of bovine serum albumin containing ovomucoid protease inhibitor. Cells were resuspended in CMF-PBS and viabil- We thank Michael Citron and David Patch for excellent ity was determined by trypan blue exclusion. The single technical assistance and Jenna McKee-Johnson and Lydia cell suspension was applied onto a discontinuous gradient Matesic for fragmentation derivatives. Some YAC clones of 35% and 60% Percoll in CMF-PBS (Pharmacia) and sub- were obtained with the assistance of the Baylor University jected to centrifugation at 800g for 10 rain. Cells were YAC screening core, C. Chinault, P.I.D.E.C. was supported collected from the interface, washed in CMF-PBS, and sus- in part by a March of Dimes Foundation predoctoral pended in 2 ml of growth medium (Dulbecco's modified award, and L.J.L. was supported by a National Science Eagle medium containing 10% horse serum, 5% fetal calf Foundation predoctoral fellowship. This work was sup- serum, 4.5 g/1 of glucose, 50 U/ml of penicillin, 50 Bg/ml of ported by U.S. Public Health Service awards HG00405 and streptomycin). Cells were plated onto poly-D-lysine (25-50 HD24605 (RHR). Bg/ml, >300,000 m.w.; Sigma) coated 35-mm Petri dishes The publication costs of this article were defrayed in for 30 min at 37°C (5% CO2) allowing astrocytes to adhere. part by payment of page charges. This article must there- The plate was then swirled gently and unattached cells fore be hereby marked "advertisement" in accordance were collected. This panning procedure was repeated and with 18 USC section 1734 solely to indicate this fact. unattached cells were washed and resuspended in growth medium. Monolayer cultures for electrophysiology and cell staining were established by plating cells on poly-D- REFERENCES lysine coated glass chips in a 24-well dish at a density of 2 x 10S/ml and incubating at 37°C for 24-48 hr before use. Breitwieser, G.E. 1991. G protein-mediated ion channel For reaggregate cultures, 5 x 104 cells were plated into one activation. Hypertension 17" 684-692. well of a 96-well plate and incubated for 12 hr at 37°C, by which time most cells had formed small aggregates that Breitwieser, G.E. and G. Szabo. 1988. 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Assessment of a mutation in the H5 domain of Girk2 as a candidate for the weaver mutation.

A E Mjaatvedt, D E Cabin, S E Cole, et al.

Genome Res. 1995 5: 453-463 Access the most recent version at doi:10.1101/gr.5.5.453

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