Shan Q et al. (2011), β Subunit M2-M3 loop conformational changes are...

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PLoS One 2011 Jan 01;611:e28105. doi: 10.1371/journal.pone.0028105.

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β Subunit M2-M3 loop conformational changes are uncoupled from α1 β glycine receptor channel gating: implications for human hereditary hyperekplexia.

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Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2-M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2-M3 loop of the β than into the α1 subunit. This suggests that the M2-M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2-M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2-M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit.

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	Figure 1. Distribution of hereditary hyperekplexia-causing mutations in the GlyR α1 and β subunits.The hereditary hyperekplexia-causing mutations that disrupt the GlyR channel function rather than block surface expression, are mapped onto the structure models [3] of GlyR α1 (A52S, E103K, R218Q, S231N, I244N, P250T, V260M, T265I, Q266H, S267N, R271L/Q, K276E and Y279C) and β (G229D) subunits.
	Figure 2. Effects of M2–M3 loop mutations on α1 β GlyR channel function.(A) The positions where mutations were introduced, K24′, V25′ and Y27′, are shown in red in a structure model of the GlyR α1 subunit (left panel). Their positions are also indicated in the amino acid sequences of the M2–M3 loops of the α1 and β subunits (right panel). (B) Example traces of currents induced by increasing glycine concentrations in the indicated receptors. (C) Averaged normalized glycine concentration–response curves for the α1WT βWT GlyR (•),α1WT βK24′A GlyR (○) and the α1K24′A βWT GlyR (▾). (D) The RM/Ws (the EC50 of the mutant α1 β GlyR divided by the EC50 of the WT α1 β GlyR) resulting from introducing the K24′A, V25′A or Y27′A mutation into the α1 (•) and β (○) subunits are shown. (** p<0.01 using the Student's t-test).
	Figure 3. VCF of α1 β GlyRs.Example current and fluorescence traces of the α19′C β and α β19′C GlyRs are shown in (A) and (C), respectively. Averaged normalized glycine concentration-response curves of current and fluorescence of the α19′Cβ and α β19′C GlyRs are shown in (B) and (D), respectively. The RF/Is (the EC50 of fluorescence divided by the EC50 of current) of the α19′C β and α β19′C GlyRs are plotted (E). (** p<0.01 using the Student's t-test).
	Figure 4. Effects of M2–M3 loop mutations on chimeric α1 β GlyR channel function.(A) The molecular identity of each chimera is schematically illustrated, with green and red denoting α1 and β subunit sequences, respectively. (B–D) The RM/Ws (the EC50 of the mutant chimeric α1 β GlyR divided by the EC50 of its relevant WT chimeric α1 β GlyR) resulting from introducing the K24′A, V25′A or Y27′A mutation. The symbol (○) represents constructs containing WT α1 and mutant β or other indicated chimeric subunit, while the symbol (•) represents constructs containing mutant α1 and WT β or other indicated chimeric subunit. (* p<0.05; ** p<0.01; n.s.d. not significantly different; using the Student's t-test).
	Figure 5. VCF of chimeric α1 β GlyRs.Averaged, normalized glycine concentration-response curves of current and fluorescence of α α-β19′C and α β-α19′C GlyRs are shown in (A) and (B), respectively. (C) The RF/Is (the EC50 of fluorescence divided by the EC50 of current) of the α19′C β, α β19′C, α α-β19′C, α β-α19′C, α αB-β19′C and α αT-β19′C GlyRs are plotted. (* p<0.05; ** p<0.01; n.s.d. not significantly different, using the Student's t-test).

References [+] :

Beato, The activation mechanism of alpha1 homomeric glycine receptors. 2004, Pubmed