Bianchi Matt T, Macdonald Robert L
Neuroscience Graduate Program, University of Michigan, Ann Arbor 48104-1687, USA.
J Physiol. 2002 Oct 1;544(Pt 1):3-18. doi: 10.1113/jphysiol.2002.020255.
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 alpha1beta3delta and alpha1beta3gamma2L GABA(A) receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mM) GABA concentration. alpha1beta3gamma2L receptors desensitized extensively (approximately 90%), with four phases (tau(1) approximately 20 ms, tau(2) approximately 400 ms, tau(3) approximately 2 s, tau(4) approximately 10 s), while alpha1beta3delta receptors desensitized slowly and less extensively (approximately 35 %), with one or two slow phases with time constants similar to tau(3) and tau(4) of alpha1beta3gamma2L receptors. To determine the structural basis of subunit-specific desensitization, delta-gamma2L chimera subunits were expressed with alpha1 and beta3 subunits. Replacing the entire N-terminus of the gamma2L subunit with delta subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of delta 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 (approximately 65 %). However, when delta subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of alpha1beta3delta receptors. The importance of TM2 sequence was confirmed by introducing gamma2 subunit TM2 residues into the delta subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing delta subunit TM2 sequence into the gamma2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct delta 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 alpha1beta3delta 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.
GABA(A)受体快速脱敏被认为决定了单个抑制性突触后电流（IPSC）的时间进程。尽管GABA(A)受体也表现出较慢的脱敏阶段，但人们对慢脱敏在调节突触功能中可能发挥的作用了解甚少。在瞬时转染的人胚肾（HEK293T）细胞中，使用饱和（1 mM）GABA浓度进行长时间（28秒）浓度阶跃时，大鼠α1β3δ和α1β3γ2L GABA(A)受体表现出不同的脱敏模式。α1β3γ2L受体广泛脱敏（约90%），有四个阶段（τ1约20毫秒，τ2约400毫秒，τ3约2秒，τ4约10秒），而α1β3δ受体脱敏缓慢且程度较低（约35%），有一两个慢阶段，时间常数与α1β3γ2L受体的τ3和τ4相似。为了确定亚基特异性脱敏的结构基础，将δ-γ2L嵌合亚基与α1和β3亚基一起表达。用δ亚基序列替换γ2L亚基的整个N端并未改变脱敏阶段的数量或程度。尽管将δ亚基序列延伸到跨膜结构域1（TM1）消除了脱敏的快速和中间成分，但两个慢阶段仍导致大量电流损失（约65%）。然而，当δ亚基序列延伸穿过TM2时，脱敏程度显著降低，与α1β3δ受体的脱敏程度无法区分。通过将γ2亚基的TM2残基引入δ亚基证实了TM2序列的重要性，这显著增加了脱敏程度，而没有引入快速或中间脱敏阶段。然而，将δ亚基的TM2序列引入γ2L亚基对脱敏速率或程度的影响最小。结果表明，不同的δ亚基结构决定了其独特的脱敏特性：缺乏快速和中间脱敏，且慢脱敏阶段贡献较小。最后，为了研究慢脱敏在突触功能中的可能作用，我们使用了脉冲序列方案。我们观察到，对于表现出广泛脱敏的受体，无论是否存在快速阶段，峰电流幅度的抑制都取决于GABA脉冲的频率和持续时间。脱敏程度最小的α1β3δ受体在脉冲序列期间表现出可忽略不计的抑制。由于在没有快速和中间阶段的情况下脱敏的受体表现出脉冲序列抑制，我们得出结论，在重复的受体激活过程中，受体可以积累到缓慢平衡的脱敏状态。这些结果可能表明，在重复激活GABA(A)受体的条件下，慢脱敏阶段对突触功能具有以前未被认识到的作用。
