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

The gamma- 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 alpha(1)beta(1)gamma(2S) receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma(2)M2 - 14' position is located in the alpha-gamma subunit interface. gamma(2) 13' faces the channel lumen. We expressed either the gamma(2)14' or the gamma(2)13' cysteine substitution mutants with alpha(1) cysteine substitution mutants between 12' and 16' and wild- type beta(1). Disulfide bonds formed spontaneously between gamma(2)14'C and both alpha(1)15'C and alpha(1)16'C and also between gamma(2)13'C and alpha(1)13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma(2)14'C and alpha(1)13' C. Disulfide bond formation rates with gamma(2)14'C were similar in the presence and absence of GABA, although the rate with alpha(1)13' C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alpha M2 would need to rotate in opposite directions by similar to 80 degrees to bring alpha(1)13' and alpha(1)15' into close proximity with gamma(2)14'. Alternatively, translational motion of alpha M2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma(2)14' 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.