Novel GLRA1 missense mutation (P250T) in dominant hyperekplexia defines an intracellular determinant of glycine receptor channel gating

Abstract

Missense mutations as well as a null allele of the human glycine receptor alpha1 subunit gene GLRA1 result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A of GLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature alpha1 polypeptide. After recombinant expression, homomeric alpha1(P250T) subunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase in Ki values. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the novel GLRA1 mutant allele P250T defines an intracellular determinant of glycine receptor channel gating.

Fig. 1.

A, Hyperekplexia allele of theGLRA1 gene in family BS. A, Pedigree of family BS. Affected individuals are indicated by filled symbols and unaffected individuals by open symbols. Only individuals volunteering for participation are included, and birth order was altered to avoid identification of affected individuals. SSCP conformers of DNA samples are depicted beneath the symbols of the corresponding individuals. Theasterisk denotes an individual displaying mild startle reactions, in addition to a pronounced fear syndrome, who was found to be homozygous for the normal allele GLRA1.B, Analysis of a normal and the hyperekplexia allele ofGLRA1. The nucleotide substitution (C → A) corresponding to position 1128 of the cDNA predicts the amino acid exchange P250T in the hyperekplexia α1 subunit allele (coding strand, gel lanes: G, A, T, C). The amino acid sequences (single letter code) encoded by the two DNA ladders and reading frombottom to top are listed next to the gel patterns.

Fig. 2.

Alignment of amino acid sequences of wild-type and mutant glycine, and GABA_A receptor subunits. Sequences represent the cytoplasmic loop between transmembrane segments M1 and M2 including the flanking regions. The last rowindicates point mutations encoded by the GLRA1 mutant alleles. Positions of transmembrane regions M1 and M2 are marked. The amino acid exchange P250T is given in bold. Sequences were retrieved from the EMBL nucleotide sequence database (http://www.ebi.ac.uk/embl.html).

Fig. 3.

Properties of the recombinant α1^P250T receptor protein. A, Western blot of membrane preparations from HEK 293. Immunostaining by monoclonal antibody mAb 4a, which specifically recognizes GlyR α subunits, produced no detectable differences between cells transfected with the wild-type (wt) or the mutated (P250T) α1 cDNA construct.B, Ligand-binding properties of recombinant GlyR α1 and α1^P250T receptors. Values present displacement of [³H]strychnine binding by unlabeled strychnine and glycine.

Fig. 4.

Whole-cell current responses of recombinant α1 and α1^P250T receptor channels. A,B, Whole-cell current responses at a holding potential of −70 mV elicited by different concentrations of glycine applied to oocytes expressing homomeric α1 (A) or α1^P250T (B) receptor channels. Bars indicate glycine applications; concentrations are millimolar. Note that the vertical scales for wild-type (A) and mutant (B) channels are different. C, Dose–response curves for wild-type (circles) and mutant (squares) receptor channels. EC₅₀ values for glycine and Hill coefficients are presented in Results.

Fig. 5.

Kinetics of recombinant α1 and α1^P250T receptor channels. A, Fast application of saturating concentrations of glycine (thick bar) to outside-out patches containing homomeric α1 channels. The 400 msec pulse of 1 mm glycine elicited outward currents at +70 mV (top trace) and inward currents at −70 mV (bottom trace). The dotted lineindicates the baseline, currents are corrected for the leak.B, Current–voltage (I–V) relation of wild-type channels. Filled symbols are theI–V relation of the peak current, andopen symbols show the I–Vrelation determined 400 msec after the peak (plateau). The current reverses at ∼0 mV. C, Same as in A for homomeric α1^P250T channels, with thebar indicating the application of 10 mmglycine. Note the different dimension of the vertical scale bar in comparison with A. D,I–V relation of mutant channels, seeB: 400 msec after exposure to glycine. E, Comparison of the desensitizing component of the current mediated by wild-type and mutant channels. Both traces are normalized to their respective peak currents. The horizontal bar indicates the application of a 1.5 sec pulse of glycine. Note the different time scale as compared with A and C. The data were low-pass filtered at f_c = 300 Hz and digitized at 1 kHz.

Fig. 6.

Single-channel properties of recombinant α1 and α1^P250T receptor channels. A, Thetop trace shows single-channel currents of homomeric wild-type channels recorded from an outside-out patch in the presence of 100 μm glycine. The bottom trace shows an experiment with a patch containing multiple homomeric mutant receptor channels, with the arrow indicating the application of 1 mm glycine. In the continued presence of glycine, the baseline (dotted line) was reached within 1 sec. Note the increase in noise after the application of glycine. The holding potential was −100 mV. For display, data were refiltered at f_c = 1 kHz. B, Nonstationary variance analysis of outside-out patches. The top panel shows 10 superimposed traces with the mean current printed ingray. The bottom panel shows the mean variance plotted versus time. C, Plot of the mean variance obtained from a total of 30 responses in this patch as a function of the mean current. The data were fitted with Equation 3 (see Materials and Methods), the obtained parameters are:i = 1.6 pS; p_open = 0.01; N = 42.

Fig. 7.

Topological predictions for the cytoplasmic M1-M2 loop of recombinant α1 and α1^P250T receptor channels. Location of transmembrane segments M1 and M2 are indicated.