Abstract
Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants alpha3L and alpha3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3-4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3-4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3-4 loops of alpha3L and alpha3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of alpha3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3-4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3-4 loop of alpha3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.
Highlights
We studied the contribution of the alternatively spliced region to domain folding and to the individual steps of ␣3 Glycine receptors (GlyRs) function
Replacement of hydroxylated residues and modification of insert length by deletion or duplication of six residues resulted in dramatic changes in current responses that could be accounted for by altered channel gating behavior
The spliced insert was identified as a novel regulatory motif of the inhibitory ␣3 glycine receptor, carrying a dual function: (i) stabilization of the secondary structure of the large cytoplasmic TM3– 4 loop of ␣3L and (ii) regulation of ion channel gating
Summary
Design and Expression of GlyR ␣3 Constructs—The large cytoplasmic TM3– 4 loop is a region of high sequence diversity between different glycine receptor subunits (Fig. 1A). The glycine receptor-specific antibody revealed double bands in the surface fraction, most likely because of different degrees of glycosylation; the ratio of these double bands, was similar for all of the constructs tested These observations indicated that alterations in the spliced insert within the TM3– 4 loop did not affect receptor biosynthesis or surface expression in the recombinant system. The EC50 and Imax values observed differed by factors of 40 and 11, respectively, compared with Ͻ2.3-fold variability of protein expression levels Those constructs that differed most in their electrophysiological properties showed similar protein expression in HEK 293 cells (Fig. 2B). Current responses of both the high activity mutant (␣3L⌬6), as well as the low activity mutants (␣3LT358A, ␣3LT358A/Y367F, and ␣3LT358A/S370A), differed markedly from the wild types (␣3L and ␣3K). 3251 Ϯ 317 2838 Ϯ 237 3938 Ϯ 452 2540 Ϯ 148 1161 Ϯ 132 2896 Ϯ 205 2738 Ϯ 142 1589 Ϯ 145 1215 Ϯ 72 3340 Ϯ 145 2451 Ϯ 275
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