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Figure 1. cGMP activation of wild-type CNGA1 channels. (A and B) Macroscopic current traces recorded in symmetric 130 mM Na+ from inside-out patches containing CNGA1 channels in the presence of 2 mM (A) or 30 µM (B) of intracellular cGMP. Currents were elicited by stepping from the â80-mV holding potential to voltages between â200 and 200 mV in 10-mV increments. Only traces every 20 mV are shown for clarity. Dotted line indicates zero current level. (C) I-V curves (mean ± SEM; n = 3â6) in the presence of 0.01 mM (filled inverted triangles), 0.03 mM (open triangles), 0.1 mM (filled circles), and 2 mM (open squares) cGMP, each normalized to the current at 200 mV. (D) Corresponding normalized G-V curves (mean ± SEM; n = 3â6). (E) Fraction of maximal current (I/Imax; mean ± SEM; n = 7) plotted against cGMP concentration for â200 mV (open circles) and 200 mV (filled circles). Black dashed curves are Hill equation fits with a common Hill coefficient (n) yielding EC50 = 66 ± 2 µM at â200 mV, EC50 = 53 ± 1 µM at 200 mV, and n = 1.48 ± 0.03. Red dashed curves are fits of Eq. 1 to both datasets simultaneously, with a single adjustable KL and all other parameters fixed (n = 1.48; K1 = 0.62, K2 = 1.04, and ZK2 = 0.25), which yield KL = 96 ± 2 µM at â200 mV and KL = 182 ± 5 µM at 200 mV.
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Figure 2. A point mutation in CNGA1's pore helix renders the channel voltage sensitive. (A) Partial crystal structure model of the Kv1.2-Kv2.1 chimeric channel (PDB 2R9R) featuring the pore helix and the selectivity filter in two diagonally opposite subunits. An oxygen in the side chain of the chimera's D375 is hydrogen-bonded to the nitrogen of W362. (BâF; Left) Macroscopic currents in 2 mM cGMP for the S350A, L351A, Y352A, S354A, and T355A mutants. Each current trace for Y352A or T355A is the average of five or three individual traces. (Right) Corresponding G-V curves (mean ± SEM; n = 4â6).
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Figure 3. The E363Q mutant channel shows extreme outward rectification at saturating cGMP concentrations. (A and B) Macroscopic currents of the E363Q mutant in 2 mM (A) and 0.1 mM (B) cGMP. (C) G-V curves (mean ± SEM; n = 5) for 2 mM (open circles) and 0.1 mM (filled circles) cGMP. (D) I/Imax (mean ± SEM; n = 3â7) plotted against cGMP concentration for 40 mV (open circles) and 200 mV (filled circles). Black dashed curves are Hill equation fits with a common Hill coefficient (n) yielding EC50 = 163 ± 10 µM at 40 mV, EC50 = 65 ± 3 µM at 200 mV, and n = 1.44 ± 0.01. Red dashed curves are fits of Eq. 1 to both datasets simultaneously, with a single adjustable KL and all other parameters fixed (n = 1.44; K1 = 5 à 10â3, K2 = 1.04, and ZK2 = 0.58), which yield KL = 166 ± 6 µM at 40 mV and KL = 67 ± 2 µM at 200 mV.
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Figure 4. Macroscopic rectification of the E363Q mutant reflects voltage gating, not asymmetric ion conduction. (A) Single-channel i-V curve (mean ± SEM; n = 4â7) of the E363Q mutant (filled circles) and the (arbitrarily scaled) macroscopic I-V curve (mean ± SEM; n = 5) in the presence of 2 mM cGMP (open circles). The dotted line through the single-channel data points is hand drawn. (B) Single-channel current amplitude of the E363Q mutant (mean ± SEM; n = 5â20) at 120 or â120 mV and in the presence or absence of 0.1 mM of extracellular Mg2+. *, P < 0.001 (one-way ANOVA). (CâF) Representative single-channel currents of the E363Q mutant recorded at 120 mV (C and D) or â120 mV (E and F) and in the absence (C and E) or presence (D and F) of 0.1 mM of extracellular Mg2+; each trace was recorded from a separate inside-out patch. (GâJ) Longer-duration macroscopic current traces recorded in 2 mM cGMP (G and H) from membrane patches of two oocytes expressing different densities of E363Q channels. Traces at â200 mV were magnified to show individual channel activity; the control traces recorded from the same patches at â200 mV and in the absence of cGMP are shown below (I and J).
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Figure 5. Gating effects of mutations at Y352 and E363 are not additive. (A) Macroscopic currents of the Y352A/E363Q double mutant in 2 mM cGMP; each trace is the average of nine individual traces. (B) Corresponding G-V curve (mean ± SEM; n = 5). (C) Residual conductance (mean ± SEM; n = 4â5) at very negative voltages of the channels containing a single E363Q (squares) or Y352A (circles) mutation, or a Y352A/ E363Q double mutation (triangles).
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Figure 6. S6 mutations and voltage gating. (A) Partial crystal structure model of the Kv1.2-Kv2.1 chimeric channel (PDB 2R9R) featuring the selectivity filter, the pore helix, and the external part of S6 in two diagonally opposite subunits. Residue numbers are those of the corresponding residues in CNGA1. Mutations at red- and gray-colored S6 residues produce voltage-gated and non-voltageâgated channels, respectively, whereas those at blue-colored ones produce nonconductive channels. (BâF) Macroscopic currents in 2 mM cGMP (left) and corresponding G-V curves (right; mean ± SEM; n = 3â7) for various CNGA1 mutants. The normalized conductance values were calculated from the current/electrochemical driving force ratio, except for those indicated by open circles in B, which were determined from isochronal tail current measurements.
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Figure 7. Distribution pattern of point mutations that produce voltage gating in the CNGA1 channel. (A) Partial sequence alignment of the CNGA1 (GI 31342442) and Kv1.2 (GI 1235594) channels for the region in which the alanine scan was performed. Mutations at red- and yellow-colored positions produce strongly and modestly voltage-gated currents, respectively, whereas those at blue-colored ones produce no or unmeasurable currents. The remaining mutations (gray-colored positions) produce non-voltageâgated currents. Residues in the dashed box are absent from the crystal structure model of the Kv1.2-Kv2.1 chimeric channel. (B) Partial crystal structure model of the Kv1.2-Kv2.1 chimeric channel (PDB 2R9R) corresponding to the entire alignment shown in A, featuring the selectivity filter, the pore helix, and S6 in one subunit. Labeled positions (numbering is that of corresponding residues in CNGA1) indicate the mutations that produce voltage-gated or partially voltage-gated channels. Structure within the dashed box, absent from the crystal structure, is arbitrary. (C) I200/Iâ200 (top) and I200/I10 (bottom) values (mean ± SEM; n = 3â7) of mutant channels. Red bars signify I200/Iâ200 > 5, yellow bars are 4 > I200/Iâ200 > 2.5, and gray bars are I200/Iâ200 for near unity in the top panel, whereas in the bottom panel they signify I200/I10 > 95, 85 > I200/I10 > 45, and I200/I10 < 35, respectively, except for the F387A mutant (arrow), which is discussed in Results.
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Figure 8. Voltage-dependent block of wild-type and mutant CNGA1 channels by intracellular PhTx. (AâF) I-V curves (mean ± SEM; n = 4â6) of wild-type (WT) and mutant channels in the presence (open circles) and absence (filled circles) of 10 µM of intracellular PhTx. (G) Fraction of current not blocked (I/Io; mean ± SEM; n = 4â6) by 10 µM PhTx for wild-type and mutant channels, plotted against membrane voltage. Curves are fits of a Boltzmann function, I/Io = 1/(1 + [PhTx]/appKdexpâZFV/RT); the fitted parameter values are listed in Table I.
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Figure 9. Effect of S4 mutations on voltage gating induced by the E363Q mutation. (AâC) Macroscopic currents recorded in 2 mM cGMP from E363Q mutant channels containing an additional substitution in S4: R272T (R2T; A), R275Q (R3Q; B), or R278Q (R4Q; C). (D) G-V curves (mean ± SEM; n = 3â8) for the E363Q mutants without (open squares) or with an additional S4 substitution: R272T (filled circles), R275Q (open triangles), or R278Q (filled inverted triangles). The four curves are superimposed.
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Figure 10. Voltage dependence of the activation of E363Q mutant channels exposed to a saturating cGMP concentration. (A) Semilogarithmic plot of relative conductance against voltage between 10 and 200 mV (taken from Fig. 3 C). The data points from 10 to 150 mV were fitted with a straight line, yielding a valence of 0.47 ± 0.01 (mean ± SEM; n = 5); the rest of the line was extrapolated. (B) Entire G-V curve (mean ± SEM; n = 5) of the E363Q mutant in the presence of 2 mM cGMP taken from Fig. 3 C. Black curve superimposed on the plot of relative conductance against voltage is a Boltzmann function fit yielding Z = 0.51 ± 0.01 and V1/2 = 240 ± 7 mV. Red dashed curve is a simulation of Eq. 2 with K1 = 5 à 10â3, K2 = 1.04, and ZK2 = 0.58 (taken from Table II). The asymptotic maximum was rescaled to one to produce the plot.
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Figure 11. Analysis of voltage gating of wild-type and E363 mutants. (AâC) Schemes representing gating of the CNGA1 channel, in which C and CL represent closed-channel states without and with cGMP bound, respectively, whereas O1 and O2 (lumped into a single open state in A) are two sequential open states in voltage-sensitive equilibrium. (D) G-V relations (mean ± SEM; n = 4â7) between 10 and 200 mV for wild-type and E363 mutant channels. Curves correspond to a simultaneous fit of Eq. 2 to the datasets with common K2. Best-fit parameters were (common) K2 = 1.04 ± 0.14 and individual K1 and ZK2 values for each mutant (listed in Table II). (E) ZK2 for each wild-type and mutant channel plotted against the channel's K1, both obtained from the simultaneous fit described above and listed in Table II. (F) G-V relations (mean ± SEM; n = 4â7) between 10 and 200 mV for WT and E363Q channels at saturating and subsaturating cGMP concentrations (taken from Figs. 1 D and 3 C). Curves are fits of Eq. 1 with adjustable parameters KL = 78 ± 4 µM and n = 1.44 ± 0.06. Fixed parameters are K2 = 1.04 (taken from D and common to all curves), K1 = 0.62 and ZK2 = 0.25 for WT, and K1 = 5 à 10â3 and ZK2 = 0.58 for E363Q channels (taken from Table II).
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