Hydroxylated residues influence desensitization behaviour of recombinant α3 glycine receptor channels

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Abstract
The human glycine receptor subunit α3 exists in two splice variants (α3K/L), with α3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3‐4 loop. Homomeric α3K glycine receptors show faster desensitization than α3L receptors. Ion channel properties were compared of α3L, α3K, and of the triple mutant α3LΔOH = α3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site‐directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch‐clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non‐desensitizing current was 68 ± 13% for α3L, 21 ± 13% for α3K, and 48 ± 16% for α3LΔOH. Desensitization time constants at saturating glycine concentration were 8.4 ± 2.8 s, 1.9 ± 2.3 s, and 2.8 ± 0.4 s, for α3L, α3K, and the triple mutant α3LΔOH, respectively. In contrast, single‐channel and whole‐cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3‐4 loop of the glycine receptor α3 subunit are crucial, but not exclusive, determinants of receptor desensitization.