The Journal of Neuroscience, February 1, 1999, 19(3):869–877 Novel GLRA1 Missense Mutation (P250T) in Dominant Hyperekplexia Defines an Intracellular Determinant of Glycine Receptor Channel Gating Brigitta Saul,1 Thomas Kuner,2 Diana Sobetzko,1,4 Wolfram Brune,3 Folker Hanefeld,4 Hans-Michael Meinck3 and Cord-Michael Becker1 1Institut fu¨ r Biochemie, Universita¨ t Erlangen-Nu¨ rnberg, D-91054 Erlangen, Germany, 2Max-Planck-Institut fu¨ r Medizinische Forschung, D-69120 Heidelberg, Germany, 3Neurologische Klinik, Universita¨ t Heidelberg, D-69120 Heidelberg, Germany, and 4Zentrum fu¨ r Kinderheilkunde, Schwerpunkt Neuropa¨ diatrie, Universita¨tGo¨ ttingen, D-37075 Go¨ ttingen, Germany Missense mutations as well as a null allele of the human glycine tion, consistent with a prolonged recovery from desensitization. receptor a1 subunit gene GLRA1 result in the neurological Apparent glycine binding was less affected, yielding an approx- disorder hyperekplexia [startle disease, stiff baby syndrome, imately fivefold increase in Ki values. Topological analysis pre- Mendelian Inheritance in Man (MIM) #149400]. In a pedigree dicts that the substitution of proline 250 leads to the loss of an showing dominant transmission of hyperekplexia, we identified angular polypeptide structure, thereby destabilizing open chan- a novel point mutation C1128A of GLRA1. This mutation en- nel conformations. Thus, the novel GLRA1 mutant allele P250T codes an amino acid substitution (P250T) in the cytoplasmic defines an intracellular determinant of glycine receptor channel loop linking transmembrane regions M1 and M2 of the mature gating. a1 polypeptide. After recombinant expression, homomeric a1 P250T subunit channels showed a strong reduction of maxi- Key words: glycine; hyperekplexia; inhibition; receptor; startle mum whole-cell chloride currents and an altered desensitiza- disease; stiff baby syndrome Strychnine-sensitive glycine receptors (GlyRs) represent a family al., 1993; Baker et al., 1994; Shiang et al., 1995; Handford et al., of ligand-gated chloride channels that exist as pentameric protein 1996; Milani et al., 1998; Nikolic et al., 1998). complexes. The GlyR isoform prevailing in brainstem and spinal Glycine binding is efficiently antagonized by the plant alkaloid cord of adult mammals is an assembly of ligand-binding a1 and strychnine, which produces both increases in muscle tone and structural b subunits (Betz, 1992; Becker, 1995; Becker and Lan- exaggerated startle responses to external stimuli (Becker, 1995). a a a gosch, 1998). In addition, 2, 3, and 4 subunit genes have been Symptoms of the human neurological disorder hyperekplexia identified in the human and rodents (Grenningloh et al., 1990; [startle disease, stiff baby syndrome, STHE, Mendelian Inheri- Kuhse et al., 1990; Kingsmore et al., 1994; Matzenbach et al., tance in Man (MIM) #14940] are reminiscent of strychnine- 1994; Nikolic et al., 1998). Mature GlyR subunit polypeptides are induced GlyR dysfunction (Tijssen et al., 1995). Affected infants thought to cross the postsynaptic membrane four times, with display exaggerated startle responses and severe muscle stiffness, transmembrane segment M2 delineating the inner wall of the which may result in fatal apnea. During the first year of life, anion pore. Determinants of ligand binding have been assigned to a muscle tone returns to normal whereas excessive startling, which the large extracellular N-terminal domain of the subunit vari- may culminate in immediate, unprotected falling, persists into ants (Betz, 1992; Breitinger and Becker, 1998). Glycinergic ago- adulthood (Ryan et al., 1994; Tijssen et al., 1995; Brune et al., nist responses also depend on amino acid residues situated within 1996). Dominant traits of hyperekplexia were found to correlate the extracellular loop linking segments M2 and M3 (Becker and to GLRA1 missense mutations affecting segment M2 and the Langosch, 1998). The human genes encoding the a1(GLRA1), extracellular M2-M3 loop (Shiang et al., 1993, 1995; Elmslie et al., a2(GLRA2), a3(GLRA3), and b subunits (GLRB) have been 1996; Milani et al., 1996). In two recessive traits, amino acid localized to the chromosomal regions 5q32, Xp21.2-p22.1, 4q33- q34, and 4q31.3, respectively (Grenningloh et al., 1990; Shiang et exchanges have been identified within segment M1 (Rees et al., 1994; Becker and Langosch, 1998). Moreover, homozygosity for a null allele demonstrated that the complete loss of GLRA1 gene Received June 25, 1998; revised Nov. 4, 1998; accepted Nov. 6, 1998. function may be tolerated in the human (Brune et al., 1996). This work was supported by the Deutsche Forschungsgemeinschaft, Bundesmin- Homologous phenotypes shown by mouse lines carrying GlyR a1 isterium fu¨r Bildung und Forschung, the European Union, and the Fonds der and b mutant alleles further support the causative role of GlyR Chemischen Industrie. We thank the members of family BS for participation in this study. Generous support by P. H. Seeburg, help with Western blotting by C. Kling, alterations in hypertonic motor disorders (Mu¨lhardt et al., 1994; invaluable discussions with H.-G. Breitinger, and a critical reading of this manu- Ryan et al., 1994; Saul et al., 1994; Kling et al., 1997). script by T. Bonk are gratefully acknowledged. We thank N. Spruston for providing Igor noise analysis macros. This study reports on a novel GLRA1 allele causing dominant Dr. Saul and Dr. Kuner contributed equally to this work. hyperekplexia. A missense mutation results in the substitution of Correspondence should be addressed to Dr. Cord-Michael Becker, Institut P250, which is located within the intracellular M1-M2 loop. fu¨r Biochemie, Universita¨t Erlangen-Nu¨rnberg, Fahrstrasse 17, D-91054 Erlangen, P250T Germany. Recombinant a1 receptors displayed moderate changes in Copyright © 1999 Society for Neuroscience 0270-6474/99/190869-09$05.00/0 agonist affinity yet dramatic alterations in chloride conductance, 870 J. Neurosci., February 1, 1999, 19(3):869–877 Saul et al. • Intracellular Determinant of Glycine Receptor Channel Gating defining proline (a1)250 as an important intracellular determi- the dose–response was monophasic (see Results), consistent with obser- nant of GlyR channel gating. vations by Taleb and Betz (1994). Current expression levels and ion selectivity. Oocytes were injected with cRNA solution (23 nl, 100 ng/ml) using a Nanoject Injector (Drummond Inc., Broomall, PA). For both a1 and a1 P250T channels, the peak currents MATERIALS AND METHODS P250T elicited by saturating glycine concentrations (a1,1mM; a1 ,10mM) GLRA1 gene structure, single-strand conformation were quantified in 10 different oocytes. As current expression levels may polymorphism analysis, and sequencing of genomic DNA vary among different batches of oocytes, the ratio Iwt/Imut was calculated The numbering scheme for GLRA1 gene structure used here follows the from the average currents determined for the same batch of oocytes. designations given by Matzenbach et al. (1994) for the murine GlyR Ratios averaged from three different batches were taken as the mean subunit genes. This is consistent with recent revisions of the GLRA1 difference in current expression between a1 P250T and a1. The reversal gene structure (Shiang et al., 1993, 1995). Genomic DNA was obtained by potential of glycine-induced whole-cell currents was determined by phenol/chloroform extraction of peripheral blood leukocytes from par- changing the voltage rampwise from 260 mV to 140 mV within 2 sec. ticipating family members. PCR amplification of GLRA1 exons and Ramps recorded in the absence of glycine were subtracted from ramps subsequent single-strand conformation polymorphism (SSCP) analysis at recorded in the presence of glycine. Two such glycine-activated ramps constant temperatures (10, 15, 20, and 25°C) was performed as described were recorded before, during, and after exposure to 50% diluted Ringer’s (Milani et al., 1998). After the detection of an informative polymor- solution. Reversal potentials were corrected for liquid junction poten- 2 2 phism, amplimers of exon 7 were cloned into pBluescript II SK (Strat- tials. Assuming that cytoplasmic Cl concentrations of Xenopus oocytes agene, La Jolla, CA) and subjected to DNA sequencing. Direct sequenc- are in the range of 100–110 mM, the Nernst equation predicts shifts of ing of genomic PCR amplimers was performed on an Applied Biosystems 12.7–15.1 mV, respectively. Prism 377 automated DNA sequencer. Kinetic parameters and current–voltage (I–V) curves. Whole-cell current 5 signals were low-pass filtered at fc 3.3 kHz and digitized at 10 kHz. The current traces (decaying part: 300 msec, starting at the peak) were fitted Generation and expression of GlyR a1 subunit constructs 5 1 2 t 5 1 to single (Eq. 1: y k0 k1 *exp( x/ )) or double (Eq. 2: y k0 k1 a 2 t 1 2 t GlyR 1 subunit cDNAs (Grenningloh et al., 1990) were cloned into a *exp( x/ 1 ) k2 *exp( x/ 2 )) exponential functions to derive the pSP64T-derived vector (Krieg and Melton, 1984). Employing the decay time constants (t). The rate of solution exchange (20–80% rise oligonucleotide-directed PCR mutagenesis method of Ho et al. (1993), time, typically 3 msec) was determined after each experiment by appli- the point mutation C1128A coding for the mutant subunit a1 P250T was cation of 10% Ringer’s solution to the recording pipette (open-tip re- introduced to the cDNA construct. For functional expression in Xenopus sponse). Voltage steps were repeatedly applied with increments of 10 mV laevis oocytes, recombinant full-length plasmids were used to generate from -100 to 1100 mV. To ascertain recovery from desensitization, synthetic capped and polyadenylated cRNA using SP6 RNA polymerase single steps were separated by 5 sec (a1) or 10 sec (a1 P250T). Glycine was (Promega, Madison, WI). The cRNAs were purified by phenol/chloro- applied for 400 msec within a voltage step lasting for 600 msec. form extraction, and ribonucleotides were eliminated using Chromaspin Single-channel analysis. Single-channel currents were low-pass filtered columns (Clontech, Palo Alto, CA). RNA contents were quantified at 10 kHz, digitized with a modified pulse-code modulation device (Sony, photometrically.