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Figure 9. Model of WT channel behavior including both Type B and Type C modes of protonation. (A) The model includes four conductance states, s1âs4. s1 is the highest conductance state (65â70 pS) and occurs when no proton is bound to the channel pore. s2 (40â45 pS) occurs when one of the independent and equivalent carboxyl-carboxylate sites is occupied by a proton, and s3 (15â20 pS) occurs when both carboxyl-carboxylate sites are occupied. s4 (â¼20â25 pS) occurs when a proton binds to the pore in the alternate, Type C, mode. The rate of proton binding is assumed to be diffusion limited for both modes of protonation. The pKa for the Type B carboxyl-carboxylate sites is taken to be 7.6, while the pKa for the Type C site is estimated to be â¼6.25. Concerted transitions can occur between s4 and s2 and are represented by the rate constants γ+ and γâ. Model parameters (explained more fully in the text): \documentclass[10pt]{article}
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\begin{equation*}{\mathrm{{\alpha}}}\;=\;6.4\;{\times}\;10^{9}{\mathrm{M}}^{-}1*{\mathrm{s}}^{-}1,\;{\mathrm{{\beta}}}\;=\;200\;{\mathrm{s}}^{-}1,\;{\mathrm{{\beta}}}^{\prime}\;=\;1,920\;{\mathrm{s}}^{-}1,\;{\mathrm{{\gamma}}}^{1}\;=\;1,920\;{\mathrm{s}}^{-}1,\;{\mathrm{and\;{\gamma}}}^{2}\;=\;200\;{\mathrm{s}}^{-}1\end{equation*}\end{document}. Gaussian noise was added to the modeled channel transitions to construct realistic amplitude histograms. The model simulations were performed using a Microsoft QuickBASIC program implemented on a personal computer. (B) Comparison of model simulations with WT channel data. (Top) Amplitude histogram calculated from the activity of a single WT channel recorded at pH 7.6 with 130 mM NaCl on both sides of the membrane (the holding voltage was â80 mV). WT channels visited current levels between the low- and middle-conductance states more often than expected, giving rise to extra density in this part of the amplitude histogram (arrow). (Middle) Amplitude histogram generated from the model with s4 omitted. (Bottom) Histogram generated from the model with s4 included. Inclusion of s4 can reproduce the extra density seen in WT amplitude histograms, suggesting that a second mode of protonation is needed to fully explain WT channel behavior.
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Figure 2. Injection of a mixture of WT and E333G CNG subunits gives rise to six types of channel behavior. (A) In addition to WT behavior (showing three distinct nonzero conductance states of 65â70, 35â40, and 15â20 pS; bottom) and pure mutant behavior (showing one nonzero conductance state of â¼25 pS; top), four hybrid types of CNG channel behavior were recorded and named Types A, B, C, and D. (Left and middle) For each channel type, a single-channel current record obtained at a holding potential of â80 mV with 130 mM NaCl, pH 7.6, on both sides of the membrane is shown alongside a corresponding amplitude histogram. Type A channels had noisy openings and gave an amplitude histogram with a low-conductance (â¼31 pS) peak and a long high-conductance tail. Type B channels showed two distinct conductance states of â¼50â65 and â¼25â40 pS. Type C channels fluctuated widely about a conductance level of â¼55 pS, giving an amplitude histogram with one broad peak and tails extending to both low and high conductances. The single-channel behavior of Type D channels looked similar to WT behavior, although the Type D amplitude histogram calculated from many openings had two broad peaks (â¼70â75 and â¼25â30 pS) rather than the three sharper peaks typical of WT channels. (Right) Average amplitude histograms calculated from all the histograms assigned to each category (pure mutant, A, B, C, D, and WT). For each hybrid channel, the group average histogram is similar to the individual example shown in the middle column, suggesting that the behavior of each of the hybrid channel types was unique. (B) Of 65 single channels recorded in patches from oocytes injected with a 2:1 mixture of WT and E333G CNG subunits, 6 mutant, 2 WT, and 57 hybrid channels were observed. Of the hybrid channels, Type A \documentclass[10pt]{article}
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\begin{equation*}(n\;=\;20)\end{equation*}\end{document} and Type B \documentclass[10pt]{article}
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\begin{equation*}(n\;=\;21)\end{equation*}\end{document} channels were found roughly twice as frequently as Types C and D channels \documentclass[10pt]{article}
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\begin{equation*}(n\;=\;8\;{\mathrm{for\;both}})\end{equation*}\end{document}.
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Figure 1. The wild-type catfish olfactory cyclic nucleotideâgated channel has three conductance states whose occupancy is governed by protonation at two independent and equivalent sites in the channel pore. (A) Single-channel record showing the activity of a WT catfish olfactory CNG channel. The current was recorded from an inside-out patch held at â80 mV and exposed to 130 mM NaCl, pH 7.6, on both sides of the membrane. Recording solutions were prepared using 2H2O to slow transitions between the three conductance levels. (B) Amplitude histogram compiled from the activity of the WT channel shown in A showing the three distinct conductance levels visited by the channel (â¼70, â¼42, and â¼18 pS). (C) Model introduced by Root and MacKinnon 1994 to explain quantitatively the behavior of the wild-type channel. In the model, four pore glutamate residues (one per subunit) combine to form two independent and equivalent protonation sites affecting ion permeation. The channel occupies the 70-pS conductance state when neither site is protonated, the 42-pS conductance state when either one of the sites is protonated, and the 18-pS conductance state when both sites are protonated.
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Figure 3. pH dependence of the hybrid CNG channels formed by coexpression of WT and E333G subunits. Amplitude histograms were compiled from single-channel currents recorded from outside-out patches with 130 mM NaCl on both sides of the membrane. Holding potential: â80 mV. The extracellular pH was varied by means of an array of capillary tubes (see materials and methods). For each channel type, all of the histograms in the figure were compiled from the same channel. Type A channels showed only a slight dependence on extracellular pH, while Types B, C, and D channels were strongly pH dependent. In particular, the Type B channel had two distinct conductance states at all pH values tested and switched smoothly from the high-conductance (â¼50â65 pS) to the low-conductance (â¼25â40 pS) state as the pH was lowered.
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Figure 4. The pH dependence of the Type B hybrid CNG channel is consistent with there being a single protonation site. (A) Type B amplitude histograms (obtained from single-channel outside-out patches at a holding potential of â80 mV with 130 mM NaCl on both sides of the membrane) were compiled at different values of extracellular pH and fitted to a sum of three Gaussian functions, corresponding to the high and low conductance states and the closed state. Amplitude histograms collected from the same channel at pHo 8.0, 7.0, and 6.0 are shown along with the three-Gaussian fit at pHo 8.0 (solid line). The relative area under the low- and high-state Gaussian peaks was used as a measure of the probability of occupancy of each state as a function of pHo. The intracellular pH was 7.6. (B) The relative area under the Gaussian functions corresponding to the high and low conductance states was plotted as a function of pH, along with a fit to a model in which the probability of occupying the low conductance state depends on protonation at a single site (dotted lines). In the model, \documentclass[10pt]{article}
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\begin{equation*}P_{{\mathrm{low\;state}}}\;=\;1/[1\;+\;10^{-}({\mathrm{pKa}}^{-\;{\mathrm{pH}})}]\end{equation*}\end{document}, where \documentclass[10pt]{article}
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\begin{equation*}{\mathrm{pK}}_{{\mathrm{a}}}\;=\;-{\mathrm{log}}[{\mathrm{K}}_{{\mathrm{a}}}]\end{equation*}\end{document} describes the binding affinity of the protonation site, and \documentclass[10pt]{article}
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\begin{equation*}P_{{\mathrm{high\;state}}}\;=\;1\;-\;P_{{\mathrm{low\;state}}}\end{equation*}\end{document}. The fit gives a pKa for the protonation site of 6.79.
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Figure 5. Expression of dimers of CNG subunits. Expression of the WT:E333G (E:G) dimer, the G:E dimer, or a 2:1 mixture of the E:E and G:G dimers gave rise to Types B and C hybrid channels, but never Types A or D channels. Two examples of each hybrid channel type seen are given along with the total number of each type observed. The examples were chosen to show the variations in single-channel behavior that we observed for each channel type: Type B channels had variable absolute single-channel amplitude (e.g., bottom left), and Type C channels rarely showed a small secondary peak in the amplitude histogram at high conductances rather than a smooth tail (seen in 2/11 Type C channels when the E:G dimer was expressed and 1/9 Type C channels when the G:E dimer was expressed; examples are shown in the top and middle rows, right). Amplitude histograms were computed from single-channel currents recorded in inside-out patches at a holding potential of â80 mV. The intra- and extracellular solutions both contained 130 mM NaCl, pH 7.6.
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Figure 6. The results of expressing CNG channel dimers can be explained if the dimers can form tetrameric channels in three ways (top): (i) symmetrically, with the A and B protomers of the dimers across the channel from each other; (ii) asymmetrically, with the A and B protomers adjacent to each other; or (iii) with the two protomers of each dimer across the channel from one another. This scheme predicts that two (and only two) hybrid species should be formed when the E:G dimer, the G:E dimer, or a mixture of the E:E and G:G dimers are expressed, as observed experimentally. The two hybrid species, Types B and C, are each predicted to contain exactly two WT and two E333G subunits, with the WT subunits oriented across the channel from each other in one case and adjacent to each other in the other.
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Figure 7. Expression of a mixture of the E:G and G:G dimers gave rise to Types A, B, and C, but never Type D, channels. Expression of a mixture of the E:G and E:E dimers gave rise to Types B, C, and D, but never Type A, channels. Examples of the measured hybrid channel types are given along with the total number of each type observed. Amplitude histograms were computed from single-channel currents recorded in inside-out patches at a holding potential of â80 mV. The intracellular and extracellular solutions contained 130 mM NaCl, pH 7.6.
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Figure 8. Identities of the six types of channels observed after expression of a mixture of WT and E333G CNG subunits, as deduced through dimer expression. Hybrid channel Types A and D can be assigned to specific structures: Type A has one WT subunit and three Glu333Gly subunits, while Type D has three WT subunits and one Glu333Gly subunit. Types B and C each have two WT and two E333G subunits, although it cannot be determined which channel type has two adjacent WT subunits (Type B/Type C, bottom) and which has two WT subunits opposite one another (Type B/Type C, top).
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