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.

Shan Q , Han L , Lynch JW .

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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 Î±1 and Î² 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 Î±1 (A52S, E103K, R218Q, S231N, I244N, P250T, V260M, T265I, Q266H, S267N, R271L/Q, K276E and Y279C) and Î² (G229D) subunits.
	Figure 2. Effects of M2âM3 loop mutations on Î±1 Î² GlyR channel function.(A) The positions where mutations were introduced, K24â², V25â² and Y27â², are shown in red in a structure model of the GlyR Î±1 subunit (left panel). Their positions are also indicated in the amino acid sequences of the M2âM3 loops of the Î±1 and Î² subunits (right panel). (B) Example traces of currents induced by increasing glycine concentrations in the indicated receptors. (C) Averaged normalized glycine concentrationâresponse curves for the Î±1WT Î²WT GlyR (â¢),Î±1WT Î²K24â²A GlyR (â) and the Î±1K24â²A Î²WT GlyR (â¾). (D) The RM/Ws (the EC50 of the mutant Î±1 Î² GlyR divided by the EC50 of the WT Î±1 Î² GlyR) resulting from introducing the K24â²A, V25â²A or Y27â²A mutation into the Î±1 (â¢) and Î² (â) subunits are shown. (** p<0.01 using the Student's t-test).
	Figure 3. VCF of Î±1 Î² GlyRs.Example current and fluorescence traces of the Î±19â²C Î² and Î± Î²19â²C GlyRs are shown in (A) and (C), respectively. Averaged normalized glycine concentration-response curves of current and fluorescence of the Î±19â²CÎ² and Î± Î²19â²C GlyRs are shown in (B) and (D), respectively. The RF/Is (the EC50 of fluorescence divided by the EC50 of current) of the Î±19â²C Î² and Î± Î²19â²C GlyRs are plotted (E). (** p<0.01 using the Student's t-test).
	Figure 4. Effects of M2âM3 loop mutations on chimeric Î±1 Î² GlyR channel function.(A) The molecular identity of each chimera is schematically illustrated, with green and red denoting Î±1 and Î² subunit sequences, respectively. (BâD) The RM/Ws (the EC50 of the mutant chimeric Î±1 Î² GlyR divided by the EC50 of its relevant WT chimeric Î±1 Î² GlyR) resulting from introducing the K24â²A, V25â²A or Y27â²A mutation. The symbol (â) represents constructs containing WT Î±1 and mutant Î² or other indicated chimeric subunit, while the symbol (â¢) represents constructs containing mutant Î±1 and WT Î² 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 Î±1 Î² GlyRs.Averaged, normalized glycine concentration-response curves of current and fluorescence of Î± Î±-Î²19â²C and Î± Î²-Î±19â²C GlyRs are shown in (A) and (B), respectively. (C) The RF/Is (the EC50 of fluorescence divided by the EC50 of current) of the Î±19â²C Î², Î± Î²19â²C, Î± Î±-Î²19â²C, Î± Î²-Î±19â²C, Î± Î±B-Î²19â²C and Î± Î±T-Î²19â²C 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