PMC

Dwell time histograms of channel activity recorded from N2a cells transiently transfected with cDNA coding for Cx43WT (A), Cx43M257 (B), Cx43H142E (C), Cx43H142K (D), and Cx43H142A (E). H142E showed significantly prolonged open time (MOT = 2.167 ± 0.148 s; N = 7, n = 276) when compared to Cx43WT (MOT = 0.137 ± 0.026 s; N = 4, n = 148). MOT recorded from M257 channels was 1.604 ± 0.150 s (N = 3, n = 194). Panels D and E depict open time histograms for two additional mutations, H142K (0.173 ± 0.010 s; N = 5, n = 450) and H142A (0.221 ± 0.010 s; N = 3, n = 331), respectively. The insets show a diagram of Cx43 topology, indicating the position of amino acid 142 (green circle; panels C–E).

Solution structure of Cx43L2-142E (amino acids 119–144). The peptide was diluted in PBS buffer (pH 5.8) and maintained at 7°C. (A) Backbone traces of the 10 Cx43L2 mutant peptide (H142E) NMR conformers that best represent the structure are aligned by superimposing the backbone atoms of residues M125–K128 (labeled in bold). (B) Cx43L2 has two α-helical domains, each containing a histidine residue (positions 126 and 142). (C) Cx43L2-H142E. The mutated histidine residue (H142E) was labeled as red. The H142E substitution not only disrupted the folding of the second α-helix, but also influenced the number of residues involved in forming the first α -helix. Helical regions for the lowest energy structure are indicated in color (red/yellow). Histidine residues in L2 (H126 and H142) are indicated in bold in panels B and C.

Single channel activity recorded from Cx43-transfected N2a cell pairs. (A) Top: recording of gap junction channel activity obtained from N2a cells expressing wild-type Cx43. All-points histogram shown at the right. Dotted lines represent current levels for the open (O), closed (C), and residual (r) states. Pulse duration: 10 s. Transjunctional voltage: +60 mV. Pulse applied every 20 s. Bottom: Histogram of amplitude of conductance transitions recorded from Cx43 wild-type channels. The histogram was best described by two Gaussian functions, centered at 80.7 pS and 104.5 pS, corresponding to the transitions between open and residual and open and closed states, respectively (N = 5, n = 383). Data as in Shibayama et al. (). (B) Top: Junctional current recording obtained from a pair of N2a cells expressing mutant H142E. Only open and closed states were apparent. Bottom: Histogram of amplitude of conductance transitions obtained from H142E-expressing cells. The red dotted line depicts the position of the Gaussian functions describing the histogram obtained from wild-type channels (data in panel A). The cluster of data points corresponding to the residual open transitions in wild-type channels was noticeably absent after the H142E mutation. The histogram was centered at 105.7 pS (N = 4, n = 339). A small peak at 49.6 pS was formed by very low frequency events (n = 43). This peak was not observed in recordings from cells expressing wild-type channels.

Ratio of amplitudes between the slow and fast components of inactivation (A) and of the steady-state G_j values versus voltage (B) of hybrid wild-type-H142 mutant junctions and homomeric wild-type junctions. Positive and negative V_j polarities in hybrid channels are defined as relative depolarization and hyperpolarization of the cell expressing Cx43WT. (A) The time course of junctional current decays induced for V_js followed a biexponential relation with a fast and slow component (indicated as τ₁ and τ₂ in ) and the mean ratio of slow/fast amplitudes (mean ± SE) decreased for increasing V_j. Note that the mutation H142E caused a large reduction in the amplitude of fast gating in the whole range of voltage explored. (B) Steady-state values of G_j were measured at the end of the 30-s pulses. The averaged G_j/V_j relationships were well described by a single Boltzmann relation for each polarity of V_j. The Boltzmann parameters of homomeric wild-type junction (•) were G_jmin = 0.36, A = 0.10 mV⁻¹, V_o = −65.7 mV for negative V_j and G_jmin = 0.24, A = 0.07 mV⁻¹, V_o = +58.5 mV for positive V_j; of hybrid RCx43wt-H142K junction (◊) were G_jmin = 0.36, A = 0.11 mV⁻¹, V_o = −62.3 mV for negative V_j and G_jmin = 0.29, A = 0.08 mV⁻¹, V_o = +57.6 mV for positive V_j; of hybrid RCx43wt-H142A junction (Δ) were G_jmin = 0.42, A = 0.11 mV⁻¹, V_o = −66.9 mV for negative V_j and G_jmin = 0.16, A = 0.07 mV⁻¹, V_o = +56.6 mV for positive V_j; and of hybrid RCx43wt-H142E junction (○) were G_jmin = 0.42, A = 0.11 mV⁻¹, V_o = −68.1 mV for negative V_j and G_jmin = 0.13, A = 0.07 mV⁻¹, V_o = +51.1 mV for positive V_j. In A and B, each point represents mean values (mean ± SE) of six pairs with <5 μS levels of electrical coupling. Asterisks indicate significant differences relative to wild-type (t-test, P < 0.05).

Voltage gating of H142 mutants. Differences of voltage gating imposed by the H142 mutations were analyzed in Xenopus oocyte pairs by forming hybrid junctions composed of wild-type and mutated hemichannels and homotypic wild-type junctions as control. (A) Sample records of junctional currents (I_j) evoked by V_j pulses of ±100 mV in 20-mV increments and of 30-s duration. Positive and negative V_j polarities are defined as relative depolarization and hyperpolarization of the cell expressing the wild-type hemichannels, which induced upward and downward I_js, respectively. Junctional currents of homotypic wild-type channels decreased in response to positive and negative V_j > 40 mV, with progressively greater and faster reductions at larger V_js. The I_j decays showed an initial closing of large amplitude and fast kinetics (arrowhead) and a second component of smaller amplitude with a slower time course observable for longer pulses (arrow). Note that the gating behavior attributable to H142A and H142E hemichannels (i.e., the outward currents) showed, respectively, a mild and pronounced reduction of the fast component of junctional currents (arrowhead) and a progressively larger slow I_j inactivation (arrow). (B) Kinetic properties. The current decays induced for V_j = +100 mV in A were well fitted by biexponential relations (τ_{1 +} τ₂). For comparison, the relative amplitude of fast (τ₁, in blue) and slow (τ₂, in red) inactivations are represented. The fast component of inactivation was significantly more prominent than the slow component for wild-type and H142K currents, whereas both components were of similar amplitude for H142A and, for H142E, the slow inactivation predominated. Mean values of time constants (mean ± SE) were τ₁= 417 ± 182 ms and τ₂ = 8.7 ± 1.3 s for wild-type, τ₁= 337 ± 253 ms and τ₂ = 10.8 ± 1.8 s for H142K, τ₁= 534 ± 325 ms and τ₂ = 8.1 ± 2.1 s for H142A, and τ₁= 643 ± 271 ms and τ₂ = 7.6 ± 1.9 s for H142E.