September 28, 2016;
Quantifying the cooperative subunit action in a multimeric membrane receptor.
In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2
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Figure 1. Concentration-activation relationships of CNGA2 concatamers.(a) Effect of an increasing number of mut-subunits. The channels are either formed by four monomers (4×wt, 4×mut) or by tetrameric concatamers (wt-wt-wt-wt, mut-wt-wt-wt, mut-mut-wt-wt, mut-mut-mut-wt, mut-mut-mut-mut). All fit parameters are provided by Supplementary Table 1a. (b) Concatamers assemble as tetrameric channels. Expression of either wt-wt-wt-wt or mut-mut-mut-mut channels alone or together with a cRNA ratio 1:1 (N = 14–21). All fit parameters are provided by Supplementary Table 1b. (c) The position of wt-subunits is irrelevant for the concatamer function. The concentration-activation relationships of four concatamers with two wt-subunits and four concatamers with one wt-subunit are plotted. The fit parameters are given in Supplementary Table 1c. The relationships for the concatamers with one wt-subunit were indistinguishable as were the relationships for the concatamers with two wt-subunits (multidimensional t-test with Holm correction, p-value = 1).
Figure 2. Global fit of concentration-activation relationships from five CNGA2 concatamers.(a) Markovian submodels describing the activation gating of five concatamers. The closed-open isomerizations, including their equilibrium constants, E0….E4, are indicated only once at the bottom. They are the same for each equally liganded state. KA1H, KA2H, KA3H, KA4H, KA1L, KA2L, KA3L, and KA4L are the equilibrium association constants for the four high and low affinity binding sites, respectively. E0 and E4 were set to 1.7 × 10−5 and 9.9 × 101 according to the single-channel experiments. (b) Global fit of the data points of the five concatamers shown in a. The values of the equilibrium constants are provided by Table 1. (c) Occupancy of the states (P) predicted by the 1wt-submodel as function of the [cGMP].