Thermodynamic profile of mutual subunit control in a heteromeric receptor

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼−3.5 to ∼−5.5 kJ ⋅ mol−1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼−9.6 to ∼−9.9 kJ ⋅ mol−1. Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed–open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

The GARP Domain of the Rod CNG Channel’s β1-subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disc Rim

Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disc-shaped membranes. Disc membranes are separated from the surrounding plasma membrane and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane where it mediates the electrical response to light. We now show that the targeted delivery of the CNG channel to the outer segment requires pre-assembly of its constituent α1 and β1 subunits and that CNGβ1 contains specific targeting information encoded within the glutamic acid-rich region of its N-terminal GARP domain. We also found that the GARP domain connects the CNG channel to photoreceptor disc rims likely through an interaction with peripherin-2 and demonstrated that this function is performed by a proline-enriched region adjacent to the GARP domain. Our data reveal fine functional specializations within the structural domains of the CNG channel and suggest that channel delivery to the outer segment is independent of peripherin-2 interactions.Significance StatementThe precise delivery and organization of signaling proteins in the ciliary outer segment organelle of photoreceptor cells is critical for light detection. We report that the CNG channel, mediating the electrical response to light in rods, contains a region within the N-terminus of its CNGβ1 subunit that encodes the outer segment targeting information for the entire channel. This targeting region is adjacent to a region that connects CNGβ1 to the rims of photoreceptor discs, likely determining the subcellular compartmentalization of the CNG channel into the outer segment plasma membrane.

Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy

Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila. Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B′-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B′-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.