|
Scheme S1.
|
|
Figure 1. A chimeric CNG channel subunit containing the BD from a modulatory (β) subunit forms a functional homomeric CNG channel. (A) Schematic of CNG channel subunit sequence showing six transmembrane segments (vertical rectangles) and a cytoplasmic COOH-terminal binding domain with two parts: the roll subdomain (a β-roll flanked by A- and B-helices, shown as an omega-shaped loop) followed by the C-helix (horizontal rectangle). Chimera X-β consists of α subunit bCNG1 sequence (thin black lines) with N-S2 region and P-loop replaced by those of the α subunit fCNG2 (thick gray), and the BD replaced by that of the β subunit rCNG5 (striped). (B, top traces) Macroscopic currents from X-β homomeric channels in an inside-out patch, elicited in response to perfused cyclic nucleotide (open bars). Washout of agonist takes several seconds (dotted outline of bars). Holding voltage is −80 mV. Gap in each current trace during perfusion represents an arbitrary interval during which the voltage-step protocol was performed. (bottom traces) Currents elicited in steady-state concentrations of cyclic nucleotide (as indicated in top traces) were measured during voltage pulses from 0 mV to potentials between +100 and −100 mV in intervals of 40 mV. Traces are averages of duplicate trials and are leak subtracted. (C) Relative currents from the patch shown in B at −100 mV in response to cAMP (closed circles) or cGMP (open circles). Lines show fits of the Hill equation with parameters (± SE) as follows: for cAMP (solid line), K1/2 = 40.3 ± 2.8 μM, h = 2.08 ± 0.16; for cGMP (dashed line), K1/2 = 57.1 ± 4.0 μM, h = 1.94 ± 0.13; Imax,cAMP/Imax,cGMP = 0.996 ± 0.021. (D) Outward rectification in the same experiments shown in C, measured by the ratio of currents at +60 and −60 mV for different agonist concentrations.
|
|
Figure 2. Single-channel recordings of homomeric X-β channels show high efficacy of opening with either cAMP or cGMP. (A1âA4) Typical 750-ms excerpts of recordings from one single-channel patch at +80 and â80 mV, with steady-state concentration of agonist as indicated; closed and open state current levels are marked by c and o, respectively.
|
|
Figure 3. X-β has normal single-channel conductance. (A1âA4) All-points current amplitude histograms compiled from the excerpts in Fig. 2 (A1âA4 respectively). Insets expand the feet of histograms in A1 (30 μM cAMP, very low Popen) and A3 (3 mM cAMP, very high Popen). With the exception of the histogram in A1 at â80 mV (materials and methods), the complete histograms (connected dots) were fitted with a sum (solid line) of Gaussian peaks (dashed lines). Arrows mark current means for multiple open states at â80 mV: O0 (unprotonated), and O+ and O++ (single and doubly protonated, respectively). Popen values from the excerpts are as follows: in 30 μM cAMP, 0.0745 at +80 mV and 0.015 at â80 mV; in 300 μM cAMP, 0.943 at +80 mV and 0.9299 at â80 mV; in 3 mM cAMP, 0.9929 at +80 mV and 0.9905 at â80 mV; and in 3 mM cGMP, 0.9523 at +80 mV and 0.9671 at â80 mV.
|
|
Figure 5. Chimera X-α is less efficiently activated by cGMP than is X-β, and exhibits desensitization at high cGMP concentrations. (A, top traces) Macroscopic current traces from homomeric X-α channels (different patch than that shown in Fig. 4 A), elicited by indicated concentrations of agonist. Channels desensitize in 30 mM cGMP and recover to the activation level of 3 mM cGMP during the slow washout of agonist (dotted portion of open bars). (bottom traces) Currents during voltage steps at indicated steady-state cGMP concentrations (tested as in Fig. 1). (B1âB3) Excerpts of single X-α channel recordings at indicated potentials and steady-state cGMP concentrations (same patch as in Fig. 4, B1âB4). Popen values from excerpt in 3 mM cGMP are 0.676 at +80 mV and 0.462 at â80 mV; values from excerpt in 30 mM cGMP (collected at steady-state after onset of desensitization) are 0.131 at +80 mV and 0.125 at â80 mV. (C) Mean doseâresponse data for X-α activation by cAMP (closed circles) and cGMP (open circles) at â100 mV, compiled from six patches expressing macroscopic currents; graph is normalized with Pmax,cAMP = 0.49, the mean from single-channel measurements (materials and methods). Lines show Hill equation fits for cAMP (solid) and cGMP (dashed; excluding data >3 mM); see Table for values of K1/2 and h. Using fitted values for Imax, the ratio Imax,cAMP/Imax,cGMP is 1.405 ± 0.034. For comparison, X-β doseâresponse curves for cAMP (solid line) and cGMP (dashed line), taken from Fig. 1C and normalized as in Fig. 4 C, are shown.
|
|
Figure 4. Chimeric CNG channel subunit X-α containing BD from the α subunit fCNG2 is less efficiently activated by cAMP than is X-β. (A) Schematic shows sequence of X-α is identical to that of X-β except that the BD sequence is that of fCNG2. (top traces) Macroscopic current traces from homomeric X-α channels elicited by the indicated concentrations of cAMP. (bottom traces) Currents during voltage steps at indicated steady-state cAMP concentrations (tested as in Fig. 1). (B1âB4) Excerpts of single X-α channel recordings at indicated potentials and steady-state cAMP concentrations (collected as in Fig. 2). Popen values from the excerpt in B4 (30 mM cAMP) are 0.778 at +80 mV and 0.646 at â80 mV. (C) Doseâresponse data (closed circles) for X-α activation by cAMP at â100 mV, from the patch shown in A. Solid line shows Hill equation fit with K1/2 = 717 ± 47 μM, h = 1.241 ± 0.051, and the graph is normalized with Pmax,cAMP at cAMP set to be 0.49, the mean from single-channel measurements. For comparison, the dashed line shows cAMP doseâresponse curve for X-β, taken from Fig. 1 C and normalized using the Pmax,cAMP of 0.98 derived from single-channel measurements.
|
|
Figure 6. Chimera X-αR/βC is poorly activated by cyclic nucleotide, whereas the complementary chimera X-βR/αC is efficiently activated by cyclic nucleotide. (A1) X-αR/βC contains the roll subdomain from fCNG2 and the C-helix from rCNG5. Single-channel recordings were made at indicated potentials with a steady-state concentration of 30 mM cAMP, with or without 5 μM Ni2+ present. (top traces) Pmax is low in absence of Ni2+ (Popen = 0.004 at +80 mV and 0.012 at â80 mV). (bottom traces) Channel opening is potentiated by Ni2+, showing only one channel is present (Popen = 0.874 at +80 mV and 0.878 at â80 mV). (A2) Mean doseâresponse data for X-αR/βC activation by cAMP (closed circles) and cGMP (open circles) at â100 mV, compiled from four patches expressing macroscopic currents (materials and methods); graph is normalized using Pmax,cAMP = 0.056, the mean from single-channel measurements. Lines show Hill equation fits with parameters (±SEM) as follows: for cAMP (solid), K1/2 = 1,570 ± 370 μM, h = 0.932 ± 0.065; for cGMP (dashed, excluding data >3 mM), K1/2 = 511 ± 37 μM, h = 1.120 ± 0.044; Imax,cAMP/Imax,cGMP = 2.85 ± 0.30. (B1) X-βR/αC contains the roll subdomain from rCNG5 and the C-helix from fCNG2. Single-channel recording excerpt (no Ni2+ present) shows high Pmax in 30 mM cAMP (Popen = 0.9878 at +80 mV and 0.9896 at â80 mV). (B2) Doseâresponse data for X-βR/αC activation by cAMP (solid circles) and cGMP (open circles) at â100 mV, from a patch with maximal current â¼400 pA; graph is normalized using Pmax,cAMP = 0.954, the mean from single-channel measurements. Lines show Hill equation fits with parameters (±SE) as follows: for cAMP (solid), K1/2 = 288 ± 20 μM, h = 1.37 ± 0.12; for cGMP (dashed), K1/2 = 95.8 ± 9.2 μM, h = 1.95 ± 0.22; Imax,cAMP/Imax,cGMP = 1.0078 ± 0.030.
|
|
Figure 7. Chimeras X-βR/αC and X-β exhibit different agonist selectivity. (A) Points plot the efficacies of the two chimeras obtained from single-channel patches from distinct oocytes. Each vertical line connects a solid and an open point plotting data for cAMP and cGMP, respectively, obtained from an individual patch. For some patches, efficacy was only measured for one agonist so only one point is plotted without a vertical line. Efficacies are plotted as ÎGsat (left axis) or equivalently as Pmax/(1 â Pmax) on a logarithmic scale (right axis). (B) Points plot the K1/2 values of the two chimeras obtained from macroscopic current patches from distinct oocytes, with cAMP and cGMP data represented as in A.
|
|
Figure 8. Analysis of cAMP activation data for chimeras using a thermodynamic linkage cycle. (A) Efficacies of cAMP activation for all single-channel patches in this study are plotted using the same axes as in Fig. 6 A; each solid point plots data obtained from an individual patch from a distinct oocyte. (B) BDs of four chimeras arranged at the corners of the linkage cycle. Wide arrows represent replacements of fCNG2 C-helix by rCNG5 C-helix; dashed arrows represent replacements of fCNG2 roll subdomain by rCNG5 subdomain. For each replacement, ÎÎGsat,cAMP (defined as the change in ÎGsat,cAMP that results from performing that replacement) is shown, in units of kJ/mol. The independence hypothesis fails because parallel sides of the cycle have different values of ÎÎGsat,cAMP.
|
