Abstract
In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.
Highlights
The pentameric ligand-gated ion channel family includes the vertebrate nicotinic, GABAA, serotonin-type A and glycine receptors, as well as prokaryotic and invertebrate receptors [1,2,3]
Our results indicate that the amino-terminal extracellular region of the β1 subunit and the regions proposed to be involved in coupling between extracellular and transmembrane domains have few residues that make significant energetic contributions to the overall receptor gating equilibrium
Several regions of the α1 subunit have been proposed to be essential for establishing the connection between the extracellular domain (ECD) and the transmembrane domain (TMD), including the "PreM1" and "TM2-link-TM3" regions [21,22]
Summary
The pentameric ligand-gated ion channel (pLGIC) family includes the vertebrate nicotinic, GABAA, serotonin-type A and glycine receptors, as well as prokaryotic and invertebrate receptors [1,2,3]. Few studies have been made of the effects on receptor activation of mutations to residues outside the channel-lining region in the non-binding subunit. In the muscle nicotinic receptor the canonical ACh-binding sites are located between the α1 subunit (principal face) and the δ and ε subunits (complementary face). Residues in the channel-lining region of the β1 subunit do make energetic contributions and the timing indicates that the change occurs later in the gating process than for homologous residues in the α1 subunit. These findings indicate that the transduction of binding energy to gating flows from the binding regions of the transmitter-binding subunits to the channel and only subsequently is transmitted to the non-binding subunit
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