Spontaneous thermal motion of the GABA_A receptor M2 channel-lining segments

The γ-aminobutyric acid type A (GABA_A) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In α₁β ₁γ₂₅ receptors the single γ subunit is flanked by an α and β subunit. The γ₂M2-14′ position is located in the α-γ subunit interface. γ ₂13′ faces the channel lumen. We expressed either the γ₂14′ or the γ₂13′ cysteine substitution mutants with α₁ cysteine substitution mutants between 12′ and 16′ and wild-type β₁. Disulfide bonds formed spontaneously between γ₂14′C and both α₁15′C and α₁16′C and also between γ₂13′C and α₁13′C. Oxidation by copper phenanthroline induced disulfide bond formation between γ₂14′C and α1₁13′C. Disulfide bond formation rates with γ₂14′C were similar in the presence and absence of GABA, although the rate with α₁13′C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, αM2 would need to rotate in opposite directions by ∼80° to bring α₁13′ and α₁15′ into close proximity with γ ₂14′. Alternatively, translational motion of αM2 would reduce the extent of rotational motion necessary to bring these two a subunit residues into close proximity with the γ₂14′ position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.