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Figure 1. Functional expression of N14K and N14Y hemichannels. Bar graphs show mean values of hemichannel (HC) currents measured in Xenopus oocytes at a holding potential of −40 mV and at the end of a 10-s voltage step to 40 mV. Measurements were obtained in 100 mM NaCl containing 0.2 mM Ca2+ (top) or 2.0 mM Ca2+ (bottom). N14K produced robust currents comparable with WT Cx26 in 0.2 mM Ca2+. Currents were substantially inhibited in 2 mM Ca2+. N14Y produced very small currents at both Ca2+ concentrations, particularly when measured at −40 mV, which were barely detectable above baseline. N14K and N14Y, each co-injected with WT Cx26 in a 1:1 ratio, showed similar differential levels of expressed currents. All oocytes were injected with the same concentration of total RNA. Measurements were obtained between 24 and 48 h, after injection. Each bar represents the mean ± SEM obtained at −40 mV and 40 mV. In 0.2 mM Ca2+: WT Cx26, n = 7; N14K, n = 13; N14Y, n = 11; N14K:WT, n = 8; N14Y:WT, n = 10. In 2 mM Ca2+: WT Cx26, n = 12; N14K, n = 9; N14Y, n = 19; N14K:WT, n = 6; N14Y:WT, n = 19.
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Figure 2. N14K and N14Y mutations produce differential shifts in voltage-dependent loop gating. Shown are representative families of currents from WT Cx26, N14K, and N14Y hemichannels at two different external Ca2+ concentrations, 0.2 mM and 2 mM. Currents were elicited using a voltage step protocol consisting of 10-s steps, from 60 to −110 mV in intervals of 10 mV, followed by a 5-s step to −110 mV; oocytes were voltage clamped to −20 mV between steps. Accompanying these currents are normalized G-V relationships at 0.2 mM and 2 mM Ca2+. Data were obtained by applying slow (600 s) voltage ramps from 40 to −100 mV from a holding potential of −20 mV. Ramps were applied to each oocyte in 0.2 and 2.0 mM Ca2+, and the calculated conductances were normalized to the maximum value measured in 0.2 mM Ca2+. N14K continued to show robust inhibition by Ca2+, but conductance remained essentially constant with hyperpolarization in 0.2 mM Ca2+. The G-V relationship for N14Y in 0.2 mM Ca2+ was shifted substantially positive along the voltage axis compared with WT Cx26, and Ca2+ appears to have a relatively modest effect. Each symbol represents the mean value. Error bars are ±SEM. WT Cx26, n = 5; N14K, n = 5; N14Y, n = 4.
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Figure 3. N14K and N14Y likely affect the stabilization of the open state for the loop gating mechanism. (A and B) Superimposition of the normalized G-V curves in 0.2 mM Ca2+ for WT and for the two mutants N14K and N14Y, expressed alone (A) and together with WT Cx26 in 1:1 ratios (B), shows the opposite shifts in activation caused by the two mutants. Data in A is the same as in Fig. 2. Error bars were omitted for clarity. N14K:WT, n = 5; N14Y:WT, n = 6; WT, n = 5. (C) Examples of deactivation kinetics for WT Cx26, N14K, and N14Y hemichannels. Cells were stepped to 40 mV for 16 s to activate hemichannels and then stepped to −100 mV (at arrows) to examine deactivation. Currents were superimposed at their peaks. N14Y currents deactivated rapidly compared with WT Cx26. N14K did not show appreciable deactivation.
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Figure 4. Single N14K hemichannel recordings show a stably open configuration. (A) Representative examples of patch clamp recordings from inside-out patches containing single WT Cx26 and N14K hemichannels. Currents shown were obtained in response to 8-s voltage ramps applied between 70 and −70 mV and leak subtracted (see Materials and methods). N14K is indistinguishable from WT Cx26 in terms of slope conductance (measured at 0 mV) and slight outward rectification. Bath and pipette solutions consisted of IPS (see Materials and methods). (B) Examples of patch clamp recordings of WT Cx26 hemichannels and three KID mutants, A40V, G45E, and N14K. Patches contained two or more active hemichannels. At the beginning of each trace, the membrane potential was stepped from 0 mV to the value indicated. WT Cx26, A40V, and G45E all showed active loop gating events throughout. N14K (red trace) showed a clear difference from the other hemichannels, essentially remaining open with no evidence of stable loop gating closures, consistent with stabilization of the open configuration for this gating mechanism. Examples of expanded views of the brief flickers (at asterisks) show them to be poorly resolved, noisy fluctuations in current that do not reach a steady level. Solid lines are the currents at 0 mV (designated I = 0). Dashed lines indicate open hemichannel current levels. The records were not leak subtracted. All currents were filtered at 1 kHz, and data were acquired at 5 kHz.
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Figure 5. The N14K mutation selectively affects the loop gating mechanism. (A) Representative cell-attached patch recording containing several N14K hemichannels. Pipettes contained IPS so that Ca2+ was buffered to submicomolar levels. The patch was stepped to voltages ranging from −90 to 60 mV for 10 s. No gating was evident with hyperpolarizing steps, but gating to subconductance states was evident on depolarizing steps characteristic of the Vj or fast gating mechanism. (B) G-V relationships at positive voltages were constructed from voltage ramps applied to patches containing one or two active hemichannels. For WT Cx26 and N14K, currents were each averaged from 50 to 60 ramps taken from eight different oocytes. The G-V curves at positive membrane voltages appear similar for WT and N14K. Error bars are ±SEM. All currents were filtered at 1 kHz, and data were acquired at 5 kHz.
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Figure 6. The N14K mutation is insensitive to pH. The bar graphs show ratios of macroscopic conductances relative to pH 8.0 in solutions adjusted to pH 7.1 and 6.5. (A) Conductances were calculated from oocytes held at −40 mV. Data are shown for WT Cx26, N14K, and N14K co-injected with WT Cx26 (N14K:WT) in ratios of 1:1 and 1:4. N14K hemichannels were insensitive to either pH 7.1 or 6.5. Hemichannel currents recorded from cells co-injected in a 1:1 ratio also were largely refractory to inhibition by pH, whereas those co-injected in a 1:4 ratio showed weakened but progressive reductions with pH 7.1 and 6.5. WT Cx26, n = 6; N14K, n = 4; N14K:WT (1:1), n = 9; N14K:WT (1:4), n = 4. (B) N14Y currents are small at negative membrane voltages, and so the effects of pH were assessed for this mutant at 40 mV. Data are shown for WT Cx26, N14K, N14Y, and each mutant co-injected with WT Cx26 in a 1:1 ratio (N14K + WT and N14Y + WT). N14K hemichannels remained insensitive to pH. N14Y showed reduced sensitivity that was intermediate between WT and N14K. WT Cx26, n = 4; N14K, n = 4; N14Y, n = 5; N14K + WT (1:1), n = 4; N14Y:WT (1:4), n = 5. Each bar represents the mean ratio ± SEM.
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Figure 7. Positions of N14 and other KID mutants in the Cx26 structure. Representation of two of six subunits (in green) around a central aqueous pore from the atomic structure of Cx26 (Protein Data Bank accession no. 2ZW3; Maeda et al., 2009). The structure is displayed using the PyMOL Molecular Graphics System (version 1.8; Schrödinger, LLC). For clarity, the Cx26 sequence shown is truncated at residue G109 near the border of the cytoplasmic TM2 extension and the cytoplasmic loop. KID mutant residues are shown as spherical renditions. The gross shape of the pore is illustrated by the dashed lines, with approximate dimensions of the pore diameter at various positions. The approximate membrane boundary is indicated. In this structure, N14 is situated in the wide cytoplasmic vestibule of the hemichannel.
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Figure 8. Substituted cysteine accessibility supports exposure of N14 to the aqueous pore. (A–C) Shown are effects of MTSEA and MTSET on macroscopic currents recorded from oocytes expressing N14C alone (A) or co-injected with WT Cx26 (B and C). Oocytes were bathed in a simple 100 mM NaCl salt solution containing the indicated Ca2+ concentrations and held at −40 mV throughout except for periodic steps to 40 mV to assess hemichannel activation at a positive voltage. (A) Responses of N14C to MTSET and MTSES. Bath application of MTSET showed little or no effect, whereas subsequent application of MTSEA produced a large induction of current. (B) The same experiment as in A, except it was performed in a cell co-injected with N14C and WT Cx26 in a 1:1 ratio. Bath application of MTSET induced an increase in current, and the subsequent action of MTSEA was attenuated. (C) In a cell co-injected with N14C and WT Cx26 in a 1:1 ratio, bath application of MTSEA induced a large current that occluded the subsequent action of MTSET, indicating that the same site is modified by both reagents and that MTSEA modifies all available sites. MTS reagents are designated as ET for MTSET and EA for MTSEA.
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Figure 9. Effects of MTS reagents on Cx26 hemichannels containing N14C subunits. (A) Bath application of the negatively charged MTSES reagent (ES) to a cell co-injected with N14C and WT Cx26 in a 1:1 ratio did not significantly affect current. Subsequent actions of MTS reagents were not affected. (B) Bath application of MTSEA biotin-XX to cells co-injected with N14C and WT Cx26 in a 1:1 ratio induced current. Subsequent actions of MTSET and MTSEA were attenuated. (C) Bar graph summarizing the effects of MTS reagents on oocytes injected with N14C and N14C + WT (1:1). Values represent the mean change in current in 0.2 mM Ca2+ after a 3-min exposure to MTSEA (yellow), MTSET (cyan), MTSES (red), and MTS biotin-XX (green). Error bars represent ±SEM; the number of oocytes tested ranged from five to nine. (D) Bar graph summarizing the effect of MTSET, MTSEA, or MTS biotin-XX application on a subsequent MTSEA application. MTSET, MTSEA, or MTS biotin-XX was applied for 3 min and followed by exposure to MTSEA for 3 min. The current after exposure to MTSEA was designated as the total current. Colored portions of each bar represent the fraction of the total current produced by the indicated MTS reagent. Co-injected oocytes show an increase in the fraction of the total current produced by MTSET or MTS biotin-XX, indicating that the N14C subunit is modified, leaving less available for subsequent MTSEA modification. Values represent mean ± SEM; the number of oocytes ranged from 6 to 10. MTS reagents were designated as ET for MTSET, EA for MTSEA, ES for MTSES, and B-XX for MTSEA biotin-XX.
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Figure 10. Effects of MTS reagents on Cx26 hemichannels containing N14C subunits on gating and unitary conductance. (A) Superimposed normalized G-V relationships of N14K hemichannels (open symbols) and N14C hemichannels modified by MTSEA (yellow symbols), MTSET (cyan symbols), and MTSEA biotin-XX (green symbols). Recordings were performed in 0.2 mM Ca2+. MTS reagents were applied for 10 min before obtaining the G-V relationships via slow (600 s) voltage ramps between 40 and −100 mV from a holding potential of −20 mV. Data points represent mean values. Error bars were omitted for clarity. N14C + MTSEA, n = 3; N14C + MTSET, n = 4; N14C + MTSEA biotin-XX, n = 5. Data for N14K is the same as in Fig. 2. (B) Superimposed patch clamp recordings from cell-attached patches containing a single WT Cx26 hemichannel and a single N14C hemichannel modified by MTSEA biotin-XX. Currents were obtained by applying 8-s voltage ramps between 70 and −70 mV and leak subtracted (see Materials and methods). The N14C hemichannel modified by MTSEA biotin-XX was only slightly reduced in conductance and exhibited a noisier and a more flickery open hemichannel current. Bath and pipette solutions consisted of IPS (see Materials and methods). Data were acquired at 5 kHz and filtered at 1 kHz. MTS reagents were designated as ET for MTSET, EA for MTSEA, and B-XX for MTSEA biotin-XX.
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Figure 11. The double mutant N14K + H100A reverses the gating and pH phenotypes caused by N14K. (A) Representation of two adjacent hemichannel subunits from the atomic structure of Cx26 (Protein Data Bank accession no. 2ZW3; Maeda et al., 2009). The structure is displayed using the PyMOL Molecular Graphics System. The N terminus (NT) of one subunit (green) comes into close proximity to the cytoplasmic extension of the TM2 domain of an adjacent subunit (cyan). The N14K mutated residue in the N terminus is shown in close association with H100 in TM2 (inset shows an expanded view). The position of the mutated residue lysine at position 14 was selected from a set of backbone-dependent rotamers. (B) Superimposed normalized G-V relationships of N14K and WT Cx26 are shown (black) together with the double mutant N14K + H100A (red symbols) and H100A (open symbols) alone. The double N14K + H100A mutation resembles WT Cx26, suggesting that N14K and H100 may interact to stabilize opening in N14K hemichannels, making them refractory to gating upon hyperpolarization. Recordings were performed in 0.2 mM Ca2+. G-V relationships were obtained by applying slow (600 s) voltage ramps between 40 and −100 mV from a holding potential of −20 mV. Each point is the mean conductance. Error bars were omitted for clarity. H100A, n = 4; N14K + 100A, n = 7. Data for N14K and WT are the same as in Fig. 2. (C) The loss of sensitivity to pH caused by the N14K mutation is also reversed in the double N14K + H100A mutant. Bar graph comparing pH responses of WT Cx26, N14K, and N14K + H100A hemichannels. Shown are data for pH 7.1 and 6.5 plotted as the ratios of the macroscopic conductances at each of these two pH values relative to pH 8.0. Conductance was calculated from oocytes held at −40 mV. Each bar represents the mean ratio ± SEM. N14K + H100A, n = 10. Data for WT Cx26 and N14K are the same as in Fig. 6.
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