Slow phases of GABAA receptor desensitization: structural determinants and possible relevance for synaptic function

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Abstract
GABAA receptor fast desensitization is thought to shape the time course of individual IPSCs. Although GABAA receptors also exhibit slower phases of desensitization, the possible role of slow desensitization in modifying synaptic function is poorly understood. In transiently transfected human embryonic kidney (HEK293T) cells, rat α1β3δ and α1β3γ2L GABAA receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mm) GABA concentration. α1β3γ2L receptors desensitized extensively (≈90 %), with four phases (τ1≈20 ms, τ2≈400 ms, τ3≈2 s, τ4≈10 s), while α1β3δ receptors desensitized slowly and less extensively (≈35 %), with one or two slow phases with time constants similar to τ3 and τ4 of α1β3γ2L receptors. To determine the structural basis of subunit-specific desensitization, δ-γ2L chimera subunits were expressed with α1 and β3 subunits. Replacing the entire N-terminus of the γ2L subunit with δ subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of δ subunit sequence into transmembrane domain 1 (TM1) abolished the fast and intermediate components of desensitization, the two slow phases still accounted for substantial current loss (≈65 %). However, when δ subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of α1β3δ receptors. The importance of TM2 sequence was confirmed by introducing γ2 subunit TM2 residues into the δ subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing δ subunit TM2 sequence into the γ2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct δ subunit structures are responsible for its unique desensitization properties: lack of fast and intermediate desensitization and small contribution of the slow phases of desensitization. Finally, to investigate the possible role of slow desensitization in synaptic function, we used a pulse train protocol. We observed inhibition of peak current amplitude that depended on the frequency and duration of GABA pulses for receptors exhibiting extensive desensitization, whether fast phases were present or not. The minimally desensitizing α1β3δ receptor exhibited negligible inhibition during pulse trains. Because receptors that desensitized without the fast and intermediate phases showed pulse train inhibition, we concluded that receptors can accumulate in slowly equilibrating desensitized states during repetitive receptor activation. These results may indicate a previously unrecognized role for the slow phases of desensitization for synaptic function under conditions of repeated GABAA receptor activation.