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Figure 1. . Residues in E1 of Cx46 near the TM1 border of Cx46 are important for the effects caused by the Cx46*32E1 substitution. (A) Sequence comparison of E1 domains of Cx46, Cx32, and Cx43. The accepted topology of a connexin subunit has four transmembrane (TM) domains, extracellular E1 and E2 loops, and cytoplasmic NH2-terminal (NT), loop (CL), and carboxy terminal (CT) domains. E42 resides at the TM1/E1 border and R76 at the E1/TM2 border. (B) Representative recordings of Cx46, Cx46*32E1, and Cx46*32E1(K49Q + S51D) hemichannel currents in Xenopus oocytes obtained with 8-s voltage ramps from â70 to +70 mV applied to cell-attached patches containing single hemichannels. The solid gray lines represent exponential fits to the open-state current. Substitution of the E1 domain of Cx32 into Cx46 (Cx46*32E1) substantially reduced unitary conductance and converted open-channel current rectification from inward to outward. Restoring the residues at positions 49 and 51 in the Cx46*32E1 chimera back to Cx46 sequence largely restored wild-type Cx46 properties.
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Figure 2. . Individual Cys substitutions from E43 through D51, with the exception of W45C, form functional hemichannels. Plotted are changes in unitary conductance (A) and magnitudes of single open-hemichannel current rectification (B). The change in unitary conductance represents the mean percentage change in the slope conductance compared with wt Cx46 measured at Vm = 0 from fitted open channel I-V relations. Open hemichannel current rectification was measured as the ratio of the currents at â70 mV and at +70 mV (see materials and methods). NC denotes nonfunctional hemichannels. Error bars represent standard deviations. For each mutant, n represents the number of separate patches examined containing single hemichannels.
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Figure 3. . SCAM using MTS-ET+ shows three residues in E1 accessible to modification in single open hemichannels. (A) Plot of the percentage change in unitary conductance for each residue after application of 1.0 mM MTS-ET+. The change in unitary conductance represents the mean percentage change in the slope conductance relative to the corresponding Cys substituted mutant measured at Vm = 0 from fitted open channel I-V relations. Access from cytoplasmic and extracellular faces of the hemichannel was examined by bath-application of MTS-ET+ to inside-out and outside-out patches, respectively. Data from excised patches of either configuration showed no differences and were pooled together with cell-attached recordings of prereacted oocytes. Large reductions in conductance were observed at three positions: E43C, G46C, and D51C. wtCx46 hemichannel currents showed no measurable change with 1 mM MTS-ET+. Error bars represent standard deviations. For each mutant, n represents the number of separate patches examined containing single hemichannels. NC denotes nonfunctional hemichannels. (B) Plot of open hemichannel current rectification in wt Cx46 (black bar) and E43C, G46C, and D51C hemichannels reacted with MTS-ET+. The degree of inward rectification decreased in all cases, most notably at D51C in which rectification turned slightly outward. Data were obtained from the same patches as in A. (C) Example of MTS-ET+ modification of a single D51C hemichannel. Shown is a current record obtained from an inside-out patch containing a single D51C hemichannel. The membrane potential was held constant at 30 mV. 1 mM MTS-ET+ was applied to the bath (first arrow). Unitary current decreased abruptly â¼20 s after application of MTS-ET+ and remained stable after washout (second arrow). An expanded view is shown of the region indicated by the black bar. (D) Comparison of single open hemichannel I-V curves of the same D51C hemichannel obtained before and 2 min after application and subsequent washout of MTS-ET+. The prereacted open hemichannel current is represented by a solid black line obtained from an exponential fit to the data from a single ramp.
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Figure 4. . SCAM using MTS-ESâ shows an increase in conductance and rectification at D51C. (A) Plot of the percentage change in unitary conductance for each residue after application of 5.0 mM MTS-ESâ. For each residue, unitary conductance represents the mean percentage change in the slope conductance relative to the corresponding Cys substituted mutant measured at Vm = 0 from fitted open channel I-V relations. As for MTS-ET+, data from inside-out and outside-out patches showed no differences and were pooled together with cell-attached recordings of prereacted oocytes. Only D51C hemichannels showed a measurable change with MTS-ESâ and that was an increase rather than a decrease in conductance. Boxed residues are those found to be modified by MTS-ET+. wtCx46 hemichannel currents showed no measurable change with 5 mM MTS-ESâ. Error bars represent standard deviations. For each mutant, n represents the number of separate patches examined containing single hemichannels. NC denotes nonfunctional hemichannels. (B and C) Examples of single hemichannel I-V relations of D51C hemichannels before and after reaction with (B) MTS-ET+ and (C) MTS-ESâ. Left panels show reacted single D51C hemichannels; black lines are fits to the open hemichannel currents from single voltage ramps. Right panels show superimposed fits of the data to D51C hemichannel currents before (solid black lines) and after (solid gray lines) reaction with MTS-ET+ or MTS-ESâ. Fits to wt Cx46 (dashed lines) open hemichannel currents are included for comparison. For each MTS reagent, the fitted open hemichannel currents shown before and after addition of reagent are from the same hemichannel. (D) Summary of data comparing unitary conductance (left panel) and open channel rectification of wt Cx46, unreacted D51C, and D51C reacted with MTS-ET+ and MTS-ESâ. The solid horizontals lines denote mean values for conductance and rectification of wt Cx46 hemichannels. Error bars represent standard deviations. For each mutant, n represents the number of separate patches examined containing single hemichannels.
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Figure 5. . Effects of modification with MTS-EA+ differ modestly from that with MTS-ET+. Examples of I-V relations of single open G46C (A) and D51C (B) hemichannel currents after application and washout of MTS-EA+ (dashed lines) or MTS-ET+ (solid lines). The lines represent fits to the data for single voltage ramps. Mean values are illustrated in Fig. 6 B.
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Figure 6. . All six subunits in Cx46 hemichannels are likely modified by MTS-ET+. (A) Example of an MTS-ET+ modification of G46C. Shown is a current record obtained from an inside-out patch containing a single G46C hemichannel. The membrane potential was held constant at 30 mV and 0.25 mM MTS-ET+ was applied to the bath (indicated by the bar). Unitary current decreased â¼25 s after MTS-ET+ application and remained stable for the remainder of the recording. An expanded view of the region indicated by the black bar shows the reduction in current occurred in a stepwise fashion (indicated by the arrows). Dashed lines indicate fully open and closed states before application of MTS-ET+. Dotted lines in the expanded view indicate currents levels ascribed to the identifiable step changes in current after application of MTS-ET+. The magnitude of the step change increased as modification progressed to completion. The opening events before the first identifiable step change appeared slightly diminished compared with the prereacted level (open state dashed line), indicating reactions may have taken place, but were small and not discernible as discrete steps within the open channel noise. (B) Summary of data comparing unitary conductance (left panels) and open-channel rectification of hemichannels in which positions 46 and 51 were modified to Lys, Arg, or Cys reacted with MTS-EA+ or MTS-ET+. wt Cx46 hemichannels are included for comparison. The solid horizontal lines denote mean values for conductance and rectification of wt Cx46 hemichannels. Error bars represent standard deviations. For each mutant, n represents the number of separate patches examined containing single hemichannels. Data for conductance and rectification were obtained from the same patches.
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Figure 7. . Reductions in unitary conductance of G46C hemichannels by MTS-ET+ results from modification of all six subunits. In all plots, counts represent the number of hemichannels whose conductance falls within the bin width shown. All currents were measured as the chord conductance at -70 mV in symmetric IPS solution. (A) Frequency distribution of single hemichannel current amplitudes of oocytes coinjected with G46C and wtCx46 cRNAs. No differences were observed between oocytes injected with 1:1 and 2:1 ratios (G46C:wt) and the data are pooled together. (B) Frequency distribution of single-hemichannel current amplitudes of oocytes coinjected with G46C and wtCx46 cRNAs in a 1:1 ratio and treated with 1 mM MTS-ET+. (C) Frequency distribution of single hemichannel current amplitudes of oocytes coinjected with G46C and wtCx46 cRNAs in a 2:1 ratio and treated with 1 mM MTS-ET+. (D) Frequency distribution of single hemichannel current amplitudes of oocytes injected only with G46C cRNA and treated with 1 mM MTS-ET+.
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Figure 8. . SCAM with MT-SET+ shows continued reactivity in TM1 and no evidence of reactivity in the topologically corresponding segment of TM3. Effects of Cys substitutions for TM1 residues E42 through R33 (A) and TM3 residues F175 through E166 (B). Solid horizontal lines indicate E1/TM1 and E2/TM3 borders according to Bennett et al. (1991). The change in unitary conductance represents the mean percentage change in the slope conductance compared with wt Cx46 measured at Vm = 0 from fitted open channel I-V relations. In general, Cys substitutions in TM1 produced increases in conductance, whereas in TM3 there was no obvious pattern. In TM1, E42C and R33C failed to form functional hemichannels and single channels were not observed from oocytes injected with V36C and I34C due to consistently low expression levels. In TM3, all Cys mutants formed functional hemichannels. Below the Cys-substitution data, SCAM results are summarized for residues D51 through R33 (A) and residues F175 through E166 (B), which span the regions shaded in black in the corresponding illustrations of connexin subunits. In TM1, large reductions in conductance with MTS-ET+ were observed at two positions, A39C and L35C. No substantial changes in conductance were observed at any position in TM3. For all mutants, the change in unitary conductance represents the mean percentage change in the slope conductance relative to the corresponding Cys substituted mutant measured at Vm = 0 from fitted open channel I-V relations. Data from excised patches of either configuration showed no differences and are pooled together with cell-attached recordings of prereacted oocytes. NC denotes nonfunctional hemichannels. ND denotes residues for which there was insufficient single-channel data for characterization.
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Figure 9. . Reactivity between L35 and G46 follows an α-helical pattern. (A) Space-fill models comparing side chains of residues composed of Lys, Arg, and Cys modified by MTS-EA+, MTS-ET+, and MTS-ESâ. The color code for atoms is H (white), C (cyan), N (blue), O (red), and S (yellow). Left and right panels show views along and down the long-axes of the side chains, respectively. (B) Space-fill α-helical representation of residues R33 through D51 of Cx46. Residues assigned to the pore are shown in color. All other residues are in green. With the exception of D51C, reacted residues fall along one face of the helix suggesting a departure from an α-helical transmembrane secondary structure in the E1/TM1 border region. (C) Helical wheel representation of residues R33 through G46 of Cx46. Reacted residues (red) reside on one side subtending an arc of â¼80 degrees. For A and B the models were generated using Hyperchem, release 3 (Hypercube, Inc.).
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Figure 10. . Schematic of an idealized single-channel current showing the proposed pattern of subunit modification that occurs with MTS-ET+. The lower dashed line indicates the fully closed state (closed) and the upper dashed line the open state before application of MTS-ET+ (open). We envision six step changes in conductance (indicated by the numbers) in the pattern shown, each representing a reaction at an individual subunit. The subunit reactions become progressively larger such that the first one is small and the last one constitutes nearly 40% of the total conductance change. The latter subunit modifications are large enough to be observed as discrete step changes in current, whereas the initial modifications are likely obscured by the open channel noise.
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