Thermodynamic profile of mutual subunit control in a heteromeric receptor

Abstract

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.

Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels

“…The global fit. In our recent analysis of the Heteromeric Allosteric (HA) model [31], we used two equal A2 subunits and an equal opening constant E x at equal degree of liganding. Herein these two limitations were overcome by extending the HA model to the more general Heteromeric Allosteric Combinational Opening (HACO) model.…”

“…For empty subunits, the fp n are unity, whereas for occupied subunits the corresponding fp n have values >1 and are specific. Hence, the 16 E x are generated by 16 [31]. The closed states of the remaining 15 models with one through four disabled binding sites were treated accordingly (S2 to S5 Figs).…”

“…We first fitted the 24 HACO model to the 16 CARs at +100 mV (S3 and S4 Tables). Compared to the simpler 17 HA model [31] (c.f. S1 and S2 Tables), the fit precision deteriorated markedly, as indicated by the relative SD (S6 Fig).…”

“…( B ) Related scheme for a heterotetrameric channel containing 16 closed (C0-C15) and 16 open states (O0-O15), requiring for the closed channel 32 binding steps ( K 1 - K 32 ) and 16 allosteric opening steps ( E 0 - E 15 ). ( C ) Cartoon scheme of the concatamer used in the analysis with the sequence N-A4-A2(1)-B1b-A2(2)-C [ 31 ]. The colors of the subunits are used throughout the article.…”

“…In an attempt to address this question, we recently constructed a full set of 16 concatameric heteromeric CNG channels in the sequence N-A4-A2-B1b-A2-C with defined wild-type and disabled, but still functional, binding sites ( Fig 1C ). Concentration-activation relationships (CARs) were subjected to a global fit analysis with 16 intimately coupled state models [ 31 ]. To simplify the analyses, all K x for the two A2 subunits and all E x at equal degree of liganding were assumed to be equal.…”