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Figure 1. Regulation of WT Cx26 and A40V and G45E mutant hemichannels by voltage and extracellular Ca2+. Representative conductance-voltage (G-V) relationships are shown expressed in Xenopus oocytes at different external Ca2+ concentrations ranging from nominal (0 added) to 2 mM obtained by applying slow (3 min) voltage ramps from 50 to â100 mV to oocytes expressing WT Cx26 (A), G45E (B), or A40V (C). The holding potential before and after each ramp was maintained at â20 mV. All conductances were normalized to the maximum value measured in 0 Ca2+. In WT and both mutant hemichannels, conductance declined strongly with hyperpolarization except in low extracellular Ca2+. Increasing Ca2+ shifted activation in the depolarizing direction in all cases. However, both mutant hemichannels appeared less sensitive to the effects of Ca2+, particularly at low Ca2+ concentrations. This reduced sensitivity to Ca2+ was strongest in A40V. In addition, G45E hemichannels showed stronger voltage-dependent closure at positive voltages (up to 50 mV).
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Figure 2. Steady-state Ca2+ response curves show that A40V hemichannels are less sensitive to external Ca2+ than WT Cx26 and G45E hemichannels. (A) Examples of recordings of WT Cx26 (top) and A40V (bottom) hemichannel currents and effects of increasing concentrations of extracellular Ca2+ (0, 0.2, 0.3, 0.5, 1.0, 2.0, and 5.0 mM). Oocytes were voltage clamped and maintained at â40 mV throughout. (B) Summary of data for WT Cx26 (â ), A40V (â), and G45E (â) compiled from several experiments such as that shown in A. Each point is the mean ± SEM of â¼20 experiments. (C) Plot showing resting membrane potentials measured in oocytes 24â48 h after RNA injection for WT Cx26 (â ), A40V (â), and G45E (â). Each point represents a measurement from an individual oocyte. Horizontal lines represent means of â25.1 ± 9.4 mV, â13.1 ± 5.5 mV, and â22.3 ± 8.0 mV for WT Cx26, A40V, and G45E, respectively. (D) The left panel shows the mean holding currents for oocytes from C when initially clamped to â40 mV. A40V-expressing oocytes, which showed the lowest resting potentials, showed the largest holding currents. The same oocytes exposed to 0 added Ca2+ (right) showed similar maximum current levels, indicating similar levels of expression.
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Figure 3. G45E hemichannels exhibit increased permeability to Ca2+. (A) Representative currents elicited by a series of voltage steps applied to oocytes expressing WT Cx26, A40V, and G45E. Oocytes were voltage clamped to â20 mV. The applied voltage step protocol consisted of 10-s steps from 60 to â100 mV in intervals of 10 mV followed by a 5-s step to â110 mV. Each series was repeated in extracellular solutions containing 0.2 mM Ca2+, 1.8 mM Ca2+, and 1.8 mM Ba2+. Currents from oocytes expressing any one of the three hemichannel types were similar in low Ca2+ (0.2 mM). Upon raising Ca2+ to 1.8 mM, a large transient inward current developed in oocytes expressing G45E when the voltage was stepped to â110 mV after depolarizing steps that activated these hemichannels. Equimolar substitution of Ca2+ with Ba2+ abolished the large transient inward current. Also, the hemichannel currents in Ba2+ resembled those in low Ca2+. (B and C) The transient current observed in oocytes expressing G45E represents a Ca2+-activated chloride current. (B) A representative recording of the reversal potential of the transient current for an oocyte bathed in 100 mM NaCl and 0.2 mM Ca2+. At various voltages (as indicated), oocytes were briefly exposed to 1.8 mM Ca2+ (indicated by the black boxes). The transient current that developed (filled in gray) reversed between â20 and â30 mV and was followed by a decay in the hemichannel current that reversed upon returning to 0.2 mM Ca2+. This protocol was repeated in an external solution in which 100 mM NaCl was replaced with a 50:50 NaCl/NaAsp. (C) Plot of peak transient current after exposure to 1.8 mM Ca2+ in 100 mM NaCl and 50:50 NaCl/NaAsp. Reversal potential shifted in accordance with the change in the extracellular Cl concentration.
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Figure 4. WT Cx26 hemichannels are also permeable to Ca2+. (A and B) Currents from oocytes expressing G45E (A) and WT Cx26 (B) recorded 24 and 48 h after injection of RNA. At 48 h, recordings are shown in 1.8 and 5.0 mM extracellular Ca2+. Voltage step protocols are the same as in Fig. 3 A. At 48 h, oocytes expressing G45E show larger hemichannel currents in 1.8 Ca2+ (compared with 24 h) along with larger Ca2+-activated Cl currents. Upon raising Ca2+ to 5 mM, G45E hemichannels continued to activate at positive voltages and to induce Ca2+-activated Cl currents. With increased expression of WT Cx26 hemichannels, Ca2+-activated Cl currents were also induced, albeit more weakly in comparison to G45E. (C) Peak chloride current is plotted as a function of the membrane conductance. Measurements were obtained from the same voltage protocol as shown in A and B). The magnitude of the chloride current was measured at the peak that developed at â110 mV after the activating voltages steps. Baseline was taken as the instantaneous current upon stepping to â110 mV that preceded the development of the large inward transient. The membrane conductance was measured at the end of the activating voltage step, the bulk of which was caused by hemichannel activation. Open symbols are data from WT Cx26, and closed symbols are from G45E. Experiments from individual oocytes are plotted as different symbol shapes. Lines represent fits (by eye) to the data for illustration purposes. n = 8 oocytes for G45E, and n = 9 oocytes for WT Cx26.
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Figure 5. G45E hemichannels exhibit a larger unitary conductance. Representative examples of patch clamp recordings from inside-out patches containing single WT Cx26 (A), A40V (B), and G45E (C) hemichannels. The single hemichannel currents shown are in response to 8-s voltage ramps between ±70 mV and are leak subtracted (see Materials and methods). In contrast to other hemichannels that generally show inward current rectification in symmetric 140 mM KCl, WT Cx26 shows slight outward rectification. A recording of a single Cx50 hemichannel in the same solutions is included for comparison in A.
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Figure 6. Cysteine substitutions at A40 and G45 produce functional hemichannels, but G45C exhibits impaired function as a result of coordination of metal ions. (AâC) Current traces from oocytes are shown expressing WT Cx26 (A), A40C (B), and G45C (C). Oocytes were held at â40 mV throughout and were exposed to a low extracellular Ca2+ (0.2 mM) solution with and without 10 µM of the heavy metal chelator TPEN. Oocytes expressing G45C show little current in 0.2 mM Ca2+ in the absence of TPEN and a large potentiation >10-fold after addition of TPEN. A40C hemichannel currents are robust in the absence of TPEN, and a small increase occurred after addition. TPEN had no effect on WT Cx26 hemichannels, which were robust upon lowering Ca2+ to 0.2 mM. (D) Summary of the effects of TPEN. Each bar represents the fold change (mean ± SEM) in current after TPEN in 0.2 mM Ca2+ (n = 9 for WT Cx26, n = 17 for A40C, and n = 51 for G45C).
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Figure 7. SCAM experiments using different MTS reagents show robust and differential effects on G45C. (A and B) The effects of MTS reagents on hemichannel currents are shown from G45C (A) and A40C (B) recorded at â40 mV. Before the recordings shown, oocytes were exposed to a solution containing 3 mM EGTA (0 Ca2+) to open hemichannels and then were exposed to the same solution containing 0.2 mM MTSET (positively charged, left) or 2.0 mM MTSES (negatively charged, right). G45C currents showed rapid and robust response to both MTS reagents that were opposite in sign, decreasing for MTSET and increasing for MTSES. Both MTS reagents decreased A40C currents, but the effects were more modest and slow in time course. (C) Summary of the percent change in current (mean ± SEM) upon application of the MTS reagent. n = 5 for WT Cx26, n = 7 for A40C, and n = 24 for G45C for MTSET. n = 7 for WT Cx26, n = 7 for A40C, and n = 23 for G45C for MTSES.
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Figure 8. Single-channel SCAM indicates that G45 is a pore-lining residue. (A) Recordings of single G45C hemichannels in excised patches. The top trace is a recording from an inside-out patch held at â40 mV. Exposure to MTSET produced a stepwise reduction in current (discernable levels indicated by dashed lines). The bottom recording is from an outside-out patch held at 40 mV. Exposure to MTSES produced a stepwise increase in current (discernable levels indicated by dashed lines). C and O depict fully closed and open states in both traces. (B) Examples of G45C hemichannel currents before and after (3 min) application of MTS reagents. The left panel shows a single G45C hemichannel recording in the absence of added reagent. The solid line is a fit to the open hemichannel current. The middle and right panels show effects of MTS reagents. Solid black lines represent fits to the open hemichannel I-V relationships before MTS application. Data and fits (gray lines) are after MTS application. (C) Same as in B for single A40C hemichannels. All currents were elicited by 8-s voltage ramps between ±70 mV. Currents were leak subtracted as described in Materials and methods.
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Figure 9. A40V and G45E mutants make functional cellâcell channels. (A) Bar graph showing junctional conductances, gj (mean ± SEM), measured in oocyte pairs injected with H2O (control, n = 10), WT Cx26 (n = 12), A40V (n = 8), and G45E (n = 7). Coupling was robust for WT Cx26 and both mutants compared with the control. Representative examples of junctional currents (left) along with steady-state Gj-Vj relationships (right) obtained from WT Cx26 (n = 4), A40V (n = 3), and G45E (n = 4) cell pairs. Gj is gj normalized to the maximum about Vj = 0. Records were taken from oocytes that were paired for shorter times to avoid series access resistance errors that accompany strong coupling. Vj steps between ±120 mV were applied in 10-mV increments. (B) Bar graph showing junctional conductances (mean ± SEM) measured in N2A cell pairs transfected with A40V and G45E (n = 42 for A40V, and n = 53 for G45E). Examples of junctional currents are shown below. Vj steps between ±120 mV were applied in 10-mV increments.
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