The α1K276E Startle Disease Mutation Reveals Multiple Intermediate States in the Gating of Glycine Receptors

Abstract

Loss-of-function mutations in human glycine receptors cause hyperekplexia, a rare inherited disease associated with an exaggerated startle response. We have studied a human disease mutation in the M2-M3 loop of the glycine receptor ␣1 subunit (K276E) using direct fitting of mechanisms to single-channel recordings with the program HJCFIT. Whole-cell recordings from HEK293 cells showed the mutation reduced the receptor glycine sensitivity. In single-channel recordings, rat homomeric ␣1 K276E receptors were barely active, even at 200 mM glycine. Coexpression of the ␤ subunit partially rescued channel function. Heteromeric mutant channels opened in brief bursts at 300 M glycine (a concentration that is near-maximal for wild type) and reached a maximum one-channel open probability of about 45% at 100 mM glycine (compared to 96% for wild type). Distributions of apparent open times contained more than one component in high glycine and, therefore, could not be described by mechanisms with only one fully liganded open state. Fits to the data were much better with mechanisms in which opening can also occur from more than one fully liganded intermediate (e.g., "primed" models). Brief pulses of glycine (ϳ3 ms, 30 mM) applied to mutant channels in outside-out patches activated currents with a slower rise time (1.5 ms) than those of wild-type channels (0.2 ms) and a much faster decay. These features were predicted reasonably well by the mechanisms obtained from fitting single-channel data. Our results show that, by slowing and impairing channel gating, the K276E mutation facilitates the detection of closed reaction intermediates in the activation pathway of glycine channels.

Allosteric and hyperekplexic mutant phenotypes investigated on an α 1 glycine receptor transmembrane structure

“…S4 D and E). This phenotype is similar to what is found in the α 1 GlyR (24,25). Likewise, V280M (26) was reported to produce a strong gain of function; we observed a phenotype similar to Q226E bearing spontaneous activity at pH 8 (Fig.…”

Correlating Structural and Energetic Changes in Glycine Receptor Activation

“…The ␣1 R19ЈQ mutation gives rise to a reduced single channel conductance and a marked decrease in glycine sensitivity when expressed as homomeric receptors (9,10). Similarly, the ␣1 K24ЈE mutation reduces glycine sensitivity in both homo-and heteromeric GlyRs, although no change in single channel conductance was reported (5,11,12). A recently identified third mutation, ␣1 Q226E (␣1 QϪ26ЈE ), located near the top of M1, gives rise to spontaneous channel opening, a reduction in single channel conductance, but no significant change in whole cell agonist sensitivity (13).of residues at 19Ј, 24Ј, and Ϫ26Ј.…”

“…This residue is highly conserved, like Arg 19 Ј, has a basic side group, and is critical to efficient channel activation in the ␣1 subunit (5,35), especially when mutated to a hyperekplexiacausing glutamic acid (11,12). Moreover, Lys 24 Ј sits on a segment (the M2-M3 linker) that is mobile enough to permit K24ЈC cross-linking between ␣1 subunits in functional GlyRs.…”

Glycine Receptor: Molecular Organization and Pathology

“…Окрім того, важливе значення має локалізація R271 -поряд із ТМ2-ТМ3-пет-лею, яка відіграє ключову роль у відкриванні каналу, забезпечуючи передавання сигналу від сайту зв'язування гліцину до сайту, відповідального за безпосереднє відкривання каналу. Ймовірно, інші мутації, локалізовані в цій петлі (K276E/Q та Y279C/S), діють за таким самим механізмом [81,84,85]. Певну роль у порушенні відкривання гліцинового ка-налу можуть відігравати такі мутації: Q266H [86], T265I, S267N [66,87], V260M [88].…”