Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments

Amal K Bera,Myles H Akabas

doi:10.1074/jbc.m504645200

Amal K Bera, Myles H Akabas

Open Access

https://doi.org/10.1074/jbc.m504645200

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Abstract

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 alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 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.

Highlights

Fast inhibitory neurotransmission in the central nervous system is largely mediated by the GABAA3 and glycine receptors [1]
The GABAA receptor closed state structure is probably similar to the Torpedo ACh receptor structure that has been solved to 4-Å resolution (10 –14)
The 4-Å resolution structure of the homologous ACh receptor provides a static view of the closed state structure but little insight into the mobility of the protein

Summary

Introduction

Fast inhibitory neurotransmission in the central nervous system is largely mediated by the GABAA3 and glycine receptors [1]. In the 4-Å resolution ACh receptor structure, the extracellular halves of the M2 segments appear loosely packed, and the narrow region of the channel, inferred to be the channel gate, is between the 9Ј and 14Ј4 levels [14]. This provides a static picture of the closed state. Motion of GABAA Receptor M2 Segments in the Closed State separation distance between the sulfhydryls, their relative orientation in the protein, and the protein’s flexibility/mobility in the region of the Cys residues. Disulfide trapping has been used to study protein mobility and proximity relationships between residues in both water-soluble and integral membrane proteins [25,26,27,28,29]

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