Wong WF , Chan KS , Michaleski MS , Haesler A , Young EC .

	Figure 1. Simple allosteric reaction scheme for bimodal agonism. Reaction scheme (Chan and Young, 2009) is illustrated by a cell-free inside-out macroscopic patch recording of bimodal CNG channels with cGMP washed into and out from the bath (solid and dashed black bars, respectively). Applying cGMP elicits inward channel current with no desensitization up to 3 mM cGMP, but with 10 mM cGMP, the steady-state macroscopic conductance (white arrow) is smaller than for 3 mM. During wash-in and wash-out of the 10-mM cGMP solution, there are brief intervals with bath cGMP concentration near 3 mM giving rise to “spikes” of maximal conductance. Details of reaction scheme notation: Closed- (C) and open- (O) channel states are equilibrating rapidly compared with agonist wash-in and wash-out. Sizes of black reaction arrows indicate the favored direction of equilibrium. Po increases with cGMP binding of up to m molecules of cGMP (pro action). Binding of the (m+1)th cGMP molecule decreases the steady-state Po (con action) to below the value obtained with m ligands. Experimental details of current trace: The previously characterized channel, X-fA2, incorporates the BD from fCNGA2 channel in a chimera with sequence from other CNG channel types (see Young et al., 2001, and Fig. 2). X-fA2 was expressed as homomers in Xenopus oocytes; patches were held at −40 mV. White arrows mark times where steady-state conductance, g, was measured with cGMP concentrations fixed at their nominal concentrations; for this example, g(10 mM cGMP)/g(3 mM cGMP) = 0.63.
	Figure 2. Extensive BD sequence substitutions preserve con action. (A) Topology of the CNG channel subunit, highlighting the BD region substituted in the X-chimera series of constructs. Alignment compares the substituted BD sequences (bounded by vertical dotted lines) originating from rCNGA4 (in X-rA4) and fCNGA2 (in X-fA2). Invariant non-BD sequences (Young et al., 2001) include bCNGA1 sequence in the C-linker and the extreme C-terminal region, respectively, before and after the BD. Dots in the sequence alignment indicate fCNGA2 residues conserved with rCNGA4; secondary structure elements are marked (α for helices, β for strands) as predicted by comparative modeling (Fig. 4). (B) X-chimera testing substitutions in the C-terminal portion of the BD. Bar represents BD sequence; below the bars, secondary structure elements are marked (letters for helices, numbers for strands, and PB for the PB cassette). Gray background color in bar represents amino acids conserved between fCNGA2 and rCNGA4; black ticks mark unconserved amino acids found in fCNGA2 but not in rCNGA4. Mean g(10 mM cGMP)/g(3 mM cGMP) values (±SD, number of patches in parentheses) include those previously reported for Construct 1 (Young et al., 2001) and for Construct 2 and X-rA4 (Chan and Young, 2009). (C) Examples of current traces for selected constructs during 10-mM cGMP pulses, with g(10 mM cGMP)/g(3 mM cGMP) as marked (based on applications of 3 mM cGMP; not depicted). Horizontal bar, 10 s; vertical bar, 1 nA.
	Figure 3. PB cassette substitution masks con action by raising gating efficacy: refutation of necessity hypothesis. (A) Dose–response relations of Construct 3 activated with cGMP (down-triangles) or cAMP (up-triangles). Points show means and error bars show SD (n ≥ 3 for each point); points are joined by straight lines without fitting. To plot Po values, conductances were normalized to the steady-state conductance measured at 3 mM cGMP (solid down-triangle) in the same patch. Then, Po at 3 mM cGMP was fixed as 0.73 based on Ni2+ potentiation (see Materials and methods). For comparison, gray curves show dose–response relations for Construct 2 activated by cGMP (solid) or cAMP (dashed); these were previously reported on a relative scale without conversion to Po values (Chan and Young, 2009), and new Ni2+ potentiation experiments in this study fix the Po at 3 mM cGMP as 0.18. All measures for both constructs in this plot were collected after completion of spontaneous run-up. (B) Example macroscopic current trace from one patch of Construct 3 before and after completion of run-up, tracking progressive increase in pro-action efficacy and masking of con action. Po scale at right is based on fixing Po = 0.73 for 3 mM cGMP after run-up. Arrows and asterisks, respectively, mark steady-state and spike currents as in Fig. 1. For this example, Psteady/Pspike = 0.80 before run-up and 0.95 after run-up.
	Figure 4. Substitution of N-terminal region of BD abolishes con action: refutation of sufficiency hypothesis. (A) Bars show substitutions in the N-terminal portion of the BD, with mean values of g(30 mM cGMP)/g(3 mM cGMP), including previously reported data for Construct 6 (Young et al., 2001). (B) Dose–response relation of Construct 8 activated with cGMP and cAMP (down-triangles and up-triangles, respectively; n ≥ 4 for all points). Conductances were converted to Po using the methods of Fig. 3, fixing Po = 0.337 at 3 mM cGMP. Black curves are Hill equation fits (see Materials and methods). Fitted parameters (±SE) are: cGMP, K1/2 = 368 ± 26 µM, h = 0.85 ± 0.04, and Pmax = 0.399 ± 0.006; cAMP, K1/2 = 520 ± 220 µM, h = 0.81 ± 0.23, and Pmax = 0.58 ± 0.05. For comparison, gray curves show X-fA2 dose–response data (Young et al., 2001) for cGMP (solid) and cAMP (dashed). (C) Model of the C-linker and BD from X-fA2 (see Results). (Left) Ribbon diagram highlights helix αA and strand β1 (orange) with the three unconserved residues (magenta) whose substitution in Construct 8 abolished con action; numbering follows fCNGA2 sequence (Fig. 1 alignment) with the substituted amino acid from rCNGA4 in parentheses. Hash marks (#) indicate three loops (αB′-αC′, PB cassette, and β4-β5), which were built without template residues. The canonical-binding site (dashed ellipse) uses key ligand contacts with R529 and T530 (green), which are universally conserved in CNG channels. (Right) Electrostatic potential mapped to molecular surface.
	Figure 5. V457E mutation abolishes con action. (A) Bar shows BD of Construct 8. Sequence alignment of the region from helix αA through strand β2 is shown for point mutants, along with their respective mean g(30 mM cGMP)/g(3 mM cGMP) ratios. Dots in alignment indicate residues conserved between fCNGA2 and rCNGA4. Amino acids mutated in each construct are circled. (B) Dose–response relation of X-fA2 V457E activated with cGMP (down-triangles; n ≥ 3 for all points). Conductances were converted to Po using the methods of Fig. 3, fixing Po = 0.359 at 3 mM cGMP. Black curve is a Hill equation fit. Fitted parameters (±SE) are: K1/2 = 350 ± 97 µM, h = 0.78 ± 0.17, and Pmax = 0.45 ± 0.03. For comparison, gray curve shows cGMP dose–response data for X-fA2 (Young et al., 2001) as in Fig. 4 B.
	Figure 6. Neighbor network around position 457 proposed by comparative modeling. All residues in this structural schematic are numbered according to fCNGA2 sequence position. Glutamate is shown in position 457, with black dashed lines indicating its interactions with first neighbors (see Discussion). Parentheses indicate residues that are not identified as first neighbors when valine occupies position 457. Gray dashed lines indicate interactions between first neighbors (black type) and second neighbors (gray type). E458 is capable of forming hydrogen bonds or polar contact with both E457 and K462 (thick dashed lines); other neighbor interactions shown are not polar.

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