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

Matt T. Bianchi* and Robert L. Macdonald†‡§

GABA_A receptor fast desensitization is thought to shape the time course of individual IPSCs. Although GABA_A 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 13 and 132L GABA_A receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mM) GABA concentration. 132L receptors desensitized extensively (~90 %), with four phases (₁ ~20 ms, ₂ ~400 ms, ₃ ~2 s, ₄ ~10 s), while 13 receptors desensitized slowly and less extensively (~35 %), with one or two slow phases with time constants similar to ₃ and ₄ of 132L receptors. To determine the structural basis of subunit-specific desensitization, -2L chimera subunits were expressed with 1 and 3 subunits. Replacing the entire N-terminus of the 2L subunit with subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of 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 subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of 13 receptors. The importance of TM2 sequence was confirmed by introducing 2 subunit TM2 residues into the subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing subunit TM2 sequence into the 2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct 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 13 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 GABA_A receptor activation.

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